Compositions and kits for enzymatic debridement and methods of using the same

ABSTRACT

A debridement enzyme for necrotic tissue is described that is not dependent upon proteolytic enzymatic activity but instead utilizes the amylase family of enzymes. The amylases (α-, β-, γ-amylase) are noted for the cleavage of the α-glycosidic bonds of polysaccharides, yielding lower molecular weight carbohydrate/sugar fragments. It has now been found that α-amylase is effective in the debridement of devitalized tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/511,912, which was filed Oct. 10, 2014, the entirety of which isincorporated herein by reference.

FIELD OF INVENTION

Enzymatic debridement of necrotic tissue in wounds is often used whensurgical debridement is unavailable or undesirable. This inventionrelates generally to the debridement of devitalized tissue by use ofenzymes, specifically enzymes that cleave carbohydrate α-1,4-glycodisiclinkages in polysaccharides, such as those of the amylase family, incontrast to the cleavage of peptide bonds in proteins.

BACKGROUND OF THE INVENTION

Wound healing is impaired by the presence of necrotic tissue, whichcontains dead cells and debris within second (deep partial thickness)and third degree (full thickness) burns (eschar) and chronic ulcers(e.g., diabetic ulcers). Necrotic tissue is devitalized tissue and itsaccumulation can result in a prolonged inflammatory response andmechanical obstruction of the wound contraction process with impededreepithelialization, preventing wounds from healing and, if needed,hindering the wound bed from taking skin grafts. Necrotic tissue issusceptible to bacterial infection, which further impedes wound healing,and may induce sepsis in severe cases. When necrotic tissue is yellow ortan colored and a stringy mass in appearance, it is often termed slough,and when the tissue is desiccated and forms a thick and leatherytexture, it is referred to as eschar. Debridement (removal) ofdevitalized tissue is needed to enhance wound closure. The debridingcomposition of this invention is thus useful in treating wounds.

Removing necrotic tissue helps to restore circulation at the wound siteas adequate oxygen delivery to the wound is critical to healing. Methodsto debride necrotic tissue include surgical debridement, enzymaticdebridement, autolytic debridement, biological debridement, andmechanical debridement, with surgical and enzymatic debridement beingmost prevalent. Surgical debridement is the fastest and most efficientmethod for debridement and is performed by trained medicalpersonnel/surgeons. However, viable tissue can be inadvertently removedbecause of a lack of clear demarcation between necrotic and viabletissue, resulting in an enlarged wound area and increased blood loss.Enzymatic debridement utilizes less training and it is generallyperformed by nursing/clinical staff, often requiring a longer durationof treatment for necrotic tissue removal. Enzymatic debridement involvesthe use of enzymes obtained from outside the body to remove non-livingtissue. Debridement enzymes cleave (cut, digest, hydrolyze) largecomponents of biological materials, particularly biomacromolecules intosmaller molecules that can dissolve and be removed. Most debridementenzymes function as proteases by cleaving protein polymer chains, and awide variety of proteases have been studied in this regard. Debridementenzymes are fast acting catalysts that produce slough of necrotictissue. Fibrinolysin is a plasma enzyme which, after being activated,attacks fibroin components in blood clots and exudates.Deoxyribonuclease is a pancreatic enzyme that specifically attacksnucleoprotein components of purulent exudates. Trypsin and chymotrypsinare nonspecific pancreatic enzymes, but can sever protein backbones atspecific amino acid residues. Enzymes from other animals includekrillase, a protease derived from Antarctic krill. Tropicalfruit-bearing plants provide a major source of debridement enzymes.Bromelain is a group of proteolytic enzymes from the stem of pineappleplants, which include three cysteine proteases. Papain is a nonspecificcysteine protease from papaya latex that cleaves a wide variety ofsubstances in necrotic tissues, including fibroin, collagen, andelastin. Ficin is a non-specific cysteine protease of similar operatingpH characteristics, and is derived from a plant latex of the ficus (fig)plant. Bacteria are also a source of debridement enzymes. Subtilisins,derived from Bacillus subtilis, are mixtures of nonspecific,water-soluble serine proteases that degrade necrotic tissues.Collagenases, which are metallopeptidases, are proteolytic enzymes thatdegrade collagen and are derived from Clostridium histolyticum.Vibriolysin is another collagen-attacking metallopeptidase, and it isderived from the bacteria Vibrio proteolyticus. Thermolysin is abacterial debridement enzyme from Bacillus thermoproteolyticus that actsnonspecifically with outstanding productivity, even at high temperature.Streptokinase, a fibrinogen activating protease from Streptococcus spp.and streptodornase, a deoxyribonuclease from hemolytic streptococci, hasalso been used in debridement (US Patent Application Number2008/0044459).

A number of non-FDA regulated topical proteolytic enzyme products weremarketed prior to 2009 for enzymatic debridement but manufacturing anddistribution were stopped by FDA because of adverse allergic eventsand/or lack of efficacy, including papain (papaya), trypsin andchymotrypsin (pancreatic enzymes), and Bacillus subtilis proteases.Currently, there are two proteolytic enzymatic debridement productsavailable in the United States, FDA approved Santyl® Ointment (Smith &Nephew), a collagenase derived from Clostridium histolyticum thatselectively digests triple helical collagen and utilizes 250 collagenaseUnits per gram of white petrolatum, and Debrase® Gel Dressing(NexoBrid), a mixture of bromelain enzymes derived from Ananas comosus(pineapple). Importantly, Santyl® Ointment cannot be used in conjunctionwith silver and iodine antimicrobials as they deactivate thecollagenase. Debrase® (MediWound Ltd.) has recently received FDA orphandrug status for eschar debridement.

U.S. Pat. No. 4,668,228 discloses a debriding tape comprising anadhesive mass on a non-gel, non-bioerodable, biocompatible occlusive orsemi-occlusive backing, where an effective amount of a debridingproteolytic enzyme in dry powdered form is situated on the adhesivesurface. When the powder is brought into contact with wound exudates theentire load of enzymes is released immediately.

In U.S. Pat. No. 5,206,026 is disclosed a film for instantaneousdelivery of a proteolytic enzyme to a wound. When exposed to aqueousliquid the film rapidly dissolves, thus releasing its contents ofenzymes simultaneously.

In U.S. Patent Application 2002/0114798 is disclosed an enzymatic wounddebrider that uses a combination of a proteolytic enzyme and ananhydrous hydrophilic poloxamer carrier.

In U.S. Pat. No. 5,120,656 there is provided a process for thedebridement of harvested bone having its periosteum intact, whichcomprises contacting the periosteum with a solution of enzyme selectedfrom the group consisting of proteolytic collagen-digesting enzyme andmixtures thereof under enzyme activity promoting conditions to loosenthe periosteum from the underlying bone surface and removing theloosened periosteum from the bone.

In U.S. Pat. No. 7,368,128, a dressing for debridement of necrotic andnon-viable tissue in a wound is described, wherein the dressingcomprises an effective amount of one or more proteolytic enzymesincorporated in a degradable polymeric material. The dressing of theinvention provides effective debridement of necrotic wounds over aprolonged period of time, as the enzymes may be released over time.

In International Patent Publication Number WO 2012/155027, wounddebridement compositions contain the proteolytic enzyme Seaprose (alsoknown as Protease S, from the fungus Aspergillus melleus). The majorenzyme in Seaprose is a semi-alkaline protease with a molecular weightaround 31 kDa. It can also contain other enzymes such as amylase, ahydrolytic enzyme that breaks down carbohydrates.

In International Patent Publication Number WO1984/002846, a topicalointment for skin surface wounds is described comprising wound-healingamounts papain, bromelain, trypsin, chymotrypsin, pancreatin, lipase,amylase, aloe extract and an organic astringent agent formulated in acarrier mixture of penetrating and non-penetrating emollient oils and apolyhydric alcohol emollient, with a plurality of protease. The ointmentis reported to reduce inflammation at the site of skin-surface woundsand acts to enhance the normal anti-inflammatory activities of the body.

In U.S. Pat. No. 6,548,556 it is reported that a proteolytic enzyme hasin part or in total the capacity to hydrolyze peptide amide bonds andthat such enzymes may also have some inherent lipolytic and/oramylolytic activity associated with the proteolytic activity, with thepreferred proteolytic enzyme being papain. Other suitable proteolyticenzymes include trypsin, chymotrypsin, streptokinase, streptodormase,ficin, pepsin, carboxypeptidase, aminopeptidase, chymopapain, bromelain,as well as other suitable enzymes, such as pancreatin, trypsin,collagenase, keratinase, carboxylase, aminopeptidase, elastase, andaspergillopeptidase. Pancreatin contains a mixture of peptidehydrolases/proteases (trypsin, chymotrypsin, elastase, carboxypeptidaseA, carboxypeptidase B), lipolytic enzymes (lipase, phospholipase A2,phospholipase B, cholinesterase, cholesterol esterase), glycosidases(α-amylase, glucosidase), and nucleases (deoxyribonuclease I,deoxyribonuclease II, ribonuclease).

Mixed debriding agent enzymes from Bacillus subtilis are described inU.S. Pat. No. 3,409,719. This enzyme product is reported to exhibitproteolytic activity against casein (phosphoprotein) and similaractivity against hemoglobin (metalloprotein). It also exhibitsamylolytic activity against gelatinized starch. It is capable of rapidlysis of fibrin, denatured collagen, elastin and exudate, which arereported to be the principal tissue protein components in the wound. Themixed enzyme compositions are shown in Table I of U.S. Pat. No.3,409,719 to contain a minimum of amylase to proteases, ranging from6.3% amylase and 93.7% proteases to 18.2% amylase and 81.8% proteases.

Severe burn wounds require surgical debridement in order to quicklyapply antimicrobials and dressings to reduce the risk of infection,(e.g., Pseudomonas aeruginosa sepsis, U.S. Pat. No. 4,772,465) and toprepare the wound bed for healing or subsequent skin grafting. Becauseof the potential of enhanced bleeding from viable tissue removal,enzymatic debridement may be preferable for removal of necrotic tissue.Wound healing is also difficult for diabetics due to cardiovascularinsufficiency and neuropathy; hence, enlarging the diabetic ulcer sizeby surgical debridement may not be desirable, and a critical need existsfor improved debridement for diabetic ulcers.

In International Patent Publication Number WO1999046368 a method fortreating wounds comprising the step of administering an effective amountof a carbohydrate-active enzyme is discussed, which is reported to havebroad-specificity for debriding burns and other wounds. Because of thehigh concentrations of glycosaminoglycans (GAGs) in skin, in burnpatients enzymes that degrade glycosaminoglycans are considered to beuseful adjuncts to burn wound debridement. Glycosaminoglycans are sugarchains consisting of repeating polymers of acidic polysaccharides,composed of building blocks of the following sugars in variouscombinations: galactose, glucose, N-acetylglucosamine,N-acetylgalactosamine, glucuronic acid, galacturonic acid and iduronicacid. It is known that carbohydrates have important roles in thefunctioning of living organisms. In addition to their metabolic roles,carbohydrates are structural components of the human body, beingcovalently attached to numerous other entities such as proteins (i.e.,as glycoproteins). Since human skin is reported to contain 10% by weightof glycosaminoglycans (which include heparin, heparan sulfate,chondroitin sulfate, hyaluronic acid (hyaluronan), dermatan sulfate, andkeratan sulfate, with chondroitin sulfate being the most prevalentglycosaminoglycan. Chondroitin sulfate also has β-1,3- andβ-1,4-linkages between predominant monomeric units. The term“carbohydrate-active enzyme” as used to specifically encompasscarbohydrate reducing enzymes, where examples of such enzymes includeglycosaminoglycan reducing enzymes such as hyaluronidases,chondroitinases, dermatanases, heparanases, heparinases and keratanases,with preferred carbohydrate-active enzymes of chondroitinases andhyaluronidases.

Infection control is also a significant unaddressed need in debridement.Burn eschar is typically dry necrotic tissue not readily infected, butsepsis can occur with second and third degree burns; therefore, havingantimicrobials in the eschar and wound bed during early stages of eschar(necrotic tissue) removal is highly desirable. In contrast to burneschar, chronic wound (diabetic ulcer) necrotic tissue appears topromote bacterial colonization, supported by the presence of water andnutrient sources from dead cells and debris. Necrotic tissue in woundsmay be associated with infection, while a majority of chronic wounds areinfected with microorganism biofilm.

A difficulty in the use of proteolytic enzymes for debridement ofnecrotic tissue is their ability for auto-digestion in aqueous solution,in addition to their potential difficulty with adverse allergic events,hypersensitivity and/or lack of efficacy. It is thus desirable toidentify a debridement composition, having a high debridement efficacyfor necrotic tissue, which is clinically simple to use, exhibits asuitable shelf-life, and is non-allergenic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of frequency versus modulus for collagen gels beforeand after treatment with different enzymes.

FIG. 2 is a graph of frequency versus complex modulus for collagen gelsbefore and after treatment with different enzymes.

FIG. 3 is a graph of weight percent boiled pig skin digested versesα-amylase concentration after one hour at room temperature.

DETAILED DESCRIPTION OF THE INVENTION

In this investigation a novel composition and approach to enzymaticdebridement is considered through the presumed cleavage of glycosidicbonds in and/or between glycosaminoglycan polysaccharides and collagenfibrils, and not by cleavage of peptide bonds of collagen by proteases,as has been done with previous enzymatic debridement products. It wasunexpectedly found that amylase, a non-proteolytic enzyme noted forcleavage of α-1,4-glycosidic bonds, such as in the catalyzed hydrolysisof starch into low molecular weight sugars, is effective in thedebridement of devitalized or necrotic tissue. These unique compositionsprovide high efficiency enzymatic debridement compositions, with asuitable shelf-life, without allergic reactions.

The debriding formulations described herein contain various types of theenzyme amylase. Amylases (α-, β-, γ-amylase) are a family of enzymesthat preferentially hydrolyze the α-glycosidic bonds of polysaccharides,yielding lower molecular weight carbohydrate/sugar fragments. In someembodiments, α-amylase is used as the amylase. Amylase occurs naturallyin humans and other mammals, and it is also is found in plants, bacteriaand fungi.

The physical behavior of skin tissue is determined primarily by anextensive extracellular matrix (ECM). The ECM is composed of aninterlocking mesh of fibrous proteins and glycosaminoglycans (GAGs). TheGAGs are carbohydrate polymers and are usually attached to ECM proteins,forming proteoglycans. In skin, type I collagen is the main proteincomponent of the ECM and the main proteoglycan components are decorinand versican. Presumably, these core proteins bind to the surface oftype I collagen fibrils, which provide mechanical strength to skin.Proteoglycan binding is required for appropriate assembly of collagenfibrils in the ECM and inhibits cleavage of collagen fibrils by matrixmetalloproteases. Proteoglycans are composed of a glycoprotein core towhich one or several GAG chains are covalently bonded. Four differentclasses of glycosaminoglycans exist in vertebrates, chondroitin sulfate,dermatan sulfate, keratan sulfate, and heparan sulfate/heparin.Hyaluronan (hyaluronic acid) is one of the chief components of theextracellular matrix, and it contributes significantly to cellproliferation and migration. However, unlike the otherglycosaminoglycans, hyaluronan does not attach to proteins to formproteoglycans but binds and retains water molecules and fills the gapsbetween collagen fibrils.

The GAGs are attached to a serine residue of the core protein by bothglycosidic β-1,4-(primarily by chondroitin sulfate, dermatan sulfate)and α-1,4-bonds (primarily by heparan sulfate/heparin), with thepredominant GAG being chondroitin sulfate. For internal linkages,hyaluronan and chondroitin sulfate are predominantly composed of β-1,3-and β-1,4-linkages, dermatan sulfate has predominantly α-1,3- andβ-1,4-linkages, and heparin/heparan sulfate have a mixture of β-1,4- andα-1,4-linkages, wherein the primary repeating unit does not contain 3 ormore α-1,4-linkages (Glycosaminoglycans and Proteoglycans,sigma.com/glycobiology), as required for cleavage by α-amylase.

A previous study discussed a method for treating wounds comprising thestep of administering an effective amount of a carbohydrate-activeenzyme, wherein such enzymes were preferentially chondroitinases,enzymes that catalyze the hydrolysis of the chondroitin chains onproteoglycans containing (1-4)-β-D- and (1-3)-β-D linkages, andhyaluronidases, enzymes that cleave hyaluronan, which contains β-1,4-and β-1,3-glycosidic bonds, with limited ability to degrade chondroitinand chondroitin sulfates (International Patent Publication Number WO1999046368).

The compositions and methods described herein pertain to the use of anon-protease hydrolytic enzyme for necrotic tissue debridement, where itwas unexpectedly found that the family of amylases, which includeα-amylase, β-amylase, and γ-amylase, was able to digest and decompose aneurotic tissue analog: boiled pig skin (U.S. Pat. No. 8,119,124), aswell as, freshly excised rat skin.

Amylase is a digestive enzyme that aids in the cleavage of bonds insugar residues in polysaccharides. It is found in two primary types inthe human body: salivary amylase and pancreatic amylase. In saliva,salivary amylase is responsible for the breakdown of starch and glycogeninto glucose, maltose, and dextrin. Pancreatic amylase further degradesstarches in the digestive system.

In some embodiments, the non-proteolytic component is greater than theproteolytic component of the debridement composition. In someembodiments, a ratio of non-proteolytic enzymes to proteolytic enzymesin the debridement composition is at least 4:1, at least 5:1, or atleast 10:1, at least 20:1 at least 40:1, at least 60:1, at least 80:1,or at least 100:1. Where the amount of proteolytic enzymes is 0 and theamount of non-proteolytic enzyme is greater than 0, the ratio is ∞:1. Insome embodiments, the debridement composition comprises less than 0.01%by weight proteolytic enzymes, or less than 0.001% by weight ofproteolytic enzymes, based on the total weight of the debridementcomposition. In some embodiments, the debridement composition comprisesup to 20% by weight of proteolytic enzyme, or up to 15% by weight ofproteolytic enzyme, or up to 10% by weight of proteolytic enzyme.

Relative to the three forms of amylase, α-amylase (also called1,4-α-D-glucan glucanohydrolase) is an endoamylase that is found in allliving organisms. It functions in a random manner by a multiple-attackmechanism on starch, glycogen and related polysaccharides andoligosaccharides with α-1,4-glycosidic linkages, ultimately yieldingglucose and maltose, as well as, larger oligosaccharides, none of whichare present in human skin. α-Amylase hydrolyzes 1,4-α-D-glucosidiclinkages in polysaccharides that contain 3 or more 1,4-α-linkedD-glucose units (Sigma Aldrich,http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/carbohydrate-analysis.html).However, α-amylase cannot hydrolyze α-1,6-bonds in glycogen andamylopectin.

β-Amylase (also called 1,4-α-D-glucan maltohydrolase) and γ-amylase(also called (amyloglucosidase, glucan 1,4-α-glucosidase, andglucoamylase) are exoamylases that are found exclusively in plants andmicroorganisms. Like α-amylase, β-amylase cannot hydrolyze 1,6-α-bonds.The β-amylase enzyme acts on the same substrates as α-amylase, but itremoves successive maltose units from the non-reducing end. γ-Amylasereleases β-D-glucose successively from the non-reducing end of thepolysaccharide chains. Various forms of γ-amylase can hydrolyze1,6-α-D-glucosidic bonds when the next bond in the sequence is a1,4-bond, and some preparations can hydrolyze 1,6- and1,3-α-D-glucosidic bonds in other polysaccharides. Combinations of theamylase enzymes are used in various preparations, such as foodproduction, sweeteners, starch saccharification, brewing and distillingindustries.

Calcium and chloride ions are essential for the activity of α-amylase.One Ca2+ is tightly bound by each enzyme molecule, facilitating theproper conformation for hydrolytic activity, and chloride ions have beenregarded as natural activators of the enzyme. Excess calcium stabilizesα-amylase towards heat. Catalytic activity is optimum at a temperaturerange between 40° C. and 45° C. and a pH of 7.0-7.5.

In this investigation, a novel composition for and approach to enzymaticdebridement by amylase is considered through the cleavage of glycosidicbonds in and/or between glycosaminoglycan polysaccharides and collagenfibrils, and not by cleavage of peptide bonds of collagen, as has beendone with previous enzymatic debridement products. The debridingformulation contains various types of the enzyme amylase. All amylases(α-, β-, γ-amylase) are a family of enzymes that preferentiallyhydrolyze the α-glycosidic bonds of polysaccharides, yielding lowermolecular weight carbohydrate/sugar fragments. α-Amylase randomlycleaves the 1,4-α-D-glycosidic linkages between the adjacent glucoseunits in the linear amylose chain of starch. A significant benefit ofα-amylase, is that it occurs naturally in humans and other mammals, andit is also found in plants, bacteria and fungi. In some embodiments, theamylase comprises a microbial amylase from bacteria or fungi. In otherembodiments, the amylase comprises a pancreatic amylase from human oranimal sources and plants.

Since amylase is not proteolytic, it does not self-digest in water, andis more stable compared to proteolytic enzymes under similar aqueousconditions. The high stability of amylase facilitates its storage in ahydrophilic formulation, which can be easily removed from a necroticwound post-debridement, unlike petrolatum-based proteolytic debridingointments.

The presence of amylase in proteolytic enzyme debridement has beenreported primarily at a much lower quantity than proteases in theseprocedures, and often is an impurity in pancreatic, proteolytic enzymes(U.S. Pat. No. 3,409,719; U.S. Pat. No. 8,540,983). Acarbohydrate-active enzyme formulation based upon chondroitinases andhyaluronidases has been reported to debride burns and other wounds(International Patent Publication Number WO1999046368). It isanticipated that these enzymes presumably would be active against theproteoglycans associated with collagen in skin, such as the predominantproteoglycan of chondroitin sulfate. No report has discussed theenzymatic debridement of necrotic tissue based upon amylase as theprincipal enzymatic debriding agent because amylase is presumed todegrade only α-1,4-glycosidic bonds such as in starch, a carbohydratenot involved in the extracellular matrix.

Starch molecules are glucose polymers linked together by the α-1,4- andα-1,6-glycosidic bonds, consisting of linear amylose and branchedamylopectin components. In order to make use of the carbon and energystored in starch, amylase, as part of the human digestive system,cleaves starch at multiple points, converting starch into smallersugars, which are eventually converted to glucose units. Because of theexistence of two types of linkages, the α-1,4- and the α-1,6-glycosidicbonds, different structures are possible for starch molecules. Anunbranched, single chain polymer with only the α-1,4-glucosidic bonds iscalled amylose. On the other hand, the presence of α-1,6-glucosidiclinkages results in the branched glucose polymer of amylopectin. Anotherclosely related compound functioning as the glucose storage in human andanimal cells is called glycogen. Glycogen has a structure similar tothat of amylopectin, except that the branches in glycogen tend to beshorter and more numerous. Neither amylose, nor amylopectin, norglycogen is believed to be present in human or animal skin as acomponent of stabilizing or interacting with collagen of theextracellular matrix.

The specificity of the bond attacked by α-amylase depends on the sourceof the enzyme. Currently, two major classes of α-amylase arecommercially produced through microbial fermentation. Based on where thecleavage occurs in the glucose polymer chain, the initial step in randomdepolymerization of starch is the splitting of large chains into varioussmaller sized segments. The breakdown of large segments drasticallyreduces the viscosity of the gelatinized starch solution, resulting inliquefaction because of the reduced viscosity of the solution. The finalstage of the depolymerization is saccharification, which resultspredominantly in the formation of monosaccharides, disaccharides, andtrisaccharides.

Because bacterial α-amylase randomly attacks only the α-1,4-bonds, itbelongs to the liquefying category. On the other hand, the fungalα-amylase belongs to the saccharifying category and attacks the secondlinkage from the nonreducing terminals (i.e., C4 end) of the straightsegment, resulting in the splitting off of two glucose units at a time,giving the disaccharide maltose. The bond breakage is thus moreextensive in saccharifying enzymes than in liquefying enzymes. Thestarch chains are literally chopped into small bits and pieces. Finally,α-amylase selectively attacks the last bond on the nonreducing terminalsand can act on both the α-1,4- and the α-1,6-glucosidic linkages at arelative rate of 1:20, resulting in the splitting off of simple glucoseunits into the solution. α-Amylase and γ-amylase may be used together toconvert starch to simple sugars.

Amylase has also been used in the cleaning of hard surfaces and fabrics,as described in International Patent Publication Number WO 2007/144856,which include all-purpose or “heavy-duty” washing agents, especiallylaundry detergents; liquid, gel or paste-form all-purpose washingagents, especially the so-called heavy-duty liquid types; liquidfine-fabric detergents; hand dishwashing agents or light dutydishwashing agents, especially those of the high-foaming type; machinedishwashing agents, including the various tablet, granular, liquid andrinse-aid types for household and institutional use; liquid cleaning anddisinfecting agents, including antibacterial hand-wash types, laundrybars, mouthwashes, denture cleaners, car or carpet shampoos, bathroomcleaners; hair shampoos and hair-rinses; shower gels and foam baths andmetal cleaners; as well as cleaning auxiliaries such as bleach additivesand “stain-stick” or pre-treat types.

Amylases are one of the main enzymes used in industry. Amylases havepotential application in a wide number of industrial processes such asfood, fermentation and pharmaceutical industries. Although α-amylasescan be obtained from plants, animals and microorganisms, enzymes fromfungal and bacterial sources have dominated applications in industry,including microorganisms of Bacillus spp. and Aspergillus spp., withmost commercial amylases being produced from bacterial sources such asBacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis,or Bacillus stearothermophilus.

The debridement compositions described herein can be formulated as aliquid, gel, hydrogel, powder, paste, ointment, lotion, emulsion, ormicroemulsion, and can be delivered to necrotic or devitalized tissue asa foam, spray, dressing, mesh, bandage, or film, wherein the latter maycontain film-forming polymers, semi-permeable or permeable polymers, ora degradable or non-degradable substrate, such as a dressing, bandage,or foamed material. The debridement composition can include one or morepharmaceutically or cosmetically acceptable carrier that is compatiblewith the enzymatic debridement composition. Examples of pharmaceuticallyor cosmetically acceptable carriers include, but are not limited to,water, normal saline (isotonic saline), Dulbecco's phosphate bufferedsaline (DPBS), phosphate buffered saline (PBS), saline solutionscontaining added calcium chloride, Ringer's solution, glycerin,propylene glycol, ethanol, isopropanol, butane-1,3-diol, liquidpoly(alkylene glycol)s (e.g., poly(ethylene glycol), polyglycerols,methyl ether-terminated poly(ethylene glycol), poly(ethyleneglycol-block-propylene glycol-block-ethylene glycol)), and water-solubleliquid silicone polyethers, water-insoluble media, such as, isopropylmyristate, isopropyl palmitate, mineral oil, dimethicone, andpetrolatum. In some embodiments, excipients can be present in an amountranging from 0% to 99.9 wt % based on the weight of the debridementcomposition.

The debridement composition can also include wetting agents, buffers,gelling agents, surfactants, chelating agents and emulsifiers. Otherexcipients could include various water-based buffers ranging in pH from5.0-7.5, silicones, polyether copolymers, vegetable and plant fats andoils, essential oils, hydrophilic and hydrophobic alcohols, vitamins,monoglycerides, and penetration enhancement esters such as laurateesters, myristate esters, palmitate esters, and stearate esters. In someembodiments, the debridement composition can be in a form selected fromliquid, gel, hydrogel, paste, cream, emulsion, and combinations thereof,and the like.

In some embodiments, the enzymatic debridement composition islyophilized to a dry powder. The lyophilized enzymatic debridementcomposition may be used in powder form, or the powder may be furtherprocessed into solutions, creams, lotions, gels, hydrogels, sprays,foams, aerosols, films, or other formulations.

In some embodiments, surfactant emulsifiers can be used to formemulsions, which facilitate compatibilization with organic solvents.Examples of organic solvents include, but are not limited to,non-stinging solvents, such as volatile silicone solvents and volatilealkanes to form water-in-oil or oil-in-water emulsions, reverseemulsions, miniemulsions (nanoemulsions), microemulsions, and reversemicroemulsions. Non-stinging volatile silicone solvents include, but arenot limited to low molecular weight polydimethylsiloxanes, such ashexamethyldisiloxane or octamethyltrisiloxane; low molecular weightcyclic siloxanes, such as hexamethylcyclotrisiloxane oroctamethylcyclotetrasiloxane; linear, branched or cyclic alkanes, suchas propane, butane, and isobutane (aerosols under pressure), pentane,hexane, heptane, octane, isooctane, and isomers thereof, petroleumdistillates, and cyclohexane; and chlorofluorocarbons, such as,trichloromonofluoromethane, dichlorodifluoromethane, anddichlorotetrafluoroethane; fluorocarbons, such as tetrafluoroethane,heptafluoropropane, 1,1-difluoroethane, pentafluoropropane,perfluoroheptane, perfluoromethylcyclohexane; hydrofluoroalkanes, suchas aerosols of 1,1,1,2,-tetrafluoroethane and1,1,1,2,3,3,3-heptafluoropropane, combinations thereof and the like; andvolatile gases under pressure, such as air, nitrous oxide, and liquidcarbon dioxide; or a mixture thereof. As will be understood, when storedunder high pressure, carbon dioxide can be present in the form of aliquid at room temperature. In some embodiments, the volatile solventcan be hexamethyldisiloxane, isooctane, and mixtures thereof. In someembodiments, the volatile solvent can be hexamethyldisiloxane. In someembodiments, solvents can be present in an amount ranging from 0% to99.9 wt % based on the weight of the debridement composition.

Water-soluble viscosity builders useful herein include, but are notlimited to, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose, guar gum, hydroxyethylguar, hydroxypropylguar,hydroxypropylmethylguar, carboxymethylguar, carboxymethylchitosan,locust bean gum, carrageenan, xanthan gum, gellan gum, Aloe vera gel,scleroglucan, schizophyllan, gum arabic, tamarind gum, poly(vinylalcohol), poly(ethylene oxide), poly(ethylene glycol), polyglycerol,poly(methyl vinyl ether), Carbomer and its salts, poly(acrylic acid) andits salts, poly(methacrylic acid) and its salts, sodiumpoly(2-acrylamido-2-methylpropanesulfonate), polyacrylamide,poly(N,N-dimethylacrylamide), poly(N-vinylacetamide),poly(N-vinylformamide), poly(2-hydroxyethyl methacrylate), poly(glycerylmethacrylate), poly(N-vinylpyrrolidone), poly(N-isopropylacrylamide) andpoly(N-vinylcaprolactam), the latter two hydrated below their LowerCritical Solution Temperatures, and the like, and combinations thereof.

In some embodiments, water-soluble polymers that are neutral in chargeand are not enzymatically degradable by amylase can be used as viscositybuilders. Examples of such viscosity builders include, but are notlimited to, poly(ethylene oxide), poly(ethylene glycol), poly(vinylalcohol), and poly(N-vinylpyrrolidone). Other viscosity builders usefulin the debridement compositions described herein include, but are notlimited to neutral polysaccharides that have β-linkages betweenmonosaccharide units, such as in methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose. Still otherviscosity builders useful in the debridement compositions describedherein include, but are not limited to, those that are anionic incharge, such as Carbomer and its salts, poly(acrylic acid) and it salts,and poly(methacrylic acid) and its salts. Still other viscosity buildersare anionic polysaccharides that have β-linkages between monosaccharideunits, such as in carboxymethylcellulose. Such viscosity builders may beemployed in amounts ranging from about 0.01 to about 50.0 weight percentof the debridement composition for preparation of viscous gels orpastes. Viscosity builders can be present in amounts ranging from 0.1 to45% by weight, from 0.5 to 25% by weight, or from 1.0 to 10.0% byweight.

Essential oils can also be added to the formulation as fragrance oraromatic agents, and/or as antimicrobial agents. Examples of essentialoils useful in the debridement compositions described herein include,but are not limited to, thymol, menthol, sandalwood, camphor, cardamom,cinnamon, jasmine, lavender, geranium, juniper, menthol, pine, lemon,rose, eucalyptus, clove, orange, oregano, mint, linalool, spearmint,peppermint, lemongrass, bergamot, citronella, cypress, nutmeg, spruce,tea tree, wintergreen (methyl salicylate), vanilla, and the like. Insome embodiments, the essential oils can be selected from thymol,sandalwood oil, wintergreen oil, eucalyptol, pine oil, and combinationsthereof. In some embodiments, essential oils can be present in an amountranging from 0% to 5 wt % based on the weight of the debridementcomposition. In some embodiments, essential oils can be present in anamount of at least 0.1 wt %, or at least 0.25 wt %, or at least 0.5 wt %based on the weight of the debridement composition.

In some embodiments, chlorophyllin, a water-soluble semi-syntheticderivative of chlorophyll, may also be used to control wound odor and toprovide anti-inflammatory properties. In some embodiments, chlorophyllincan be present in an amount ranging from 0% to 5 wt % based on theweight of the debridement composition. In some embodiments,chlorophyllin can be present in an amount of at least 0.1 wt %, or atleast 0.25 wt %, or at least 0.5 wt % based on the weight of thedebridement composition.

A keratolytic agent can also be added to the debridement composition toaid in digesting the eschar tissue. For example, the keratolytic agentcan promote the softening and peeling of the epidermis. Keratolyticagents useful in the debridement compositions as described hereininclude, but are not limited to, urea, salicylic acid andα-hydroxyacids, such as lactic acid, glycolic acid, and citric acid. Forexample, urea can be used to help remove dead tissue in necrotic woundsto help wound healing (U.S. Pat. No. 8,754,045). In some embodiments,the keratolytic agent can be present in an amount ranging from 0% to 15wt % based on the weight of the debridement composition. In someembodiments, essential oils can be present in an amount of at least 0.1wt %, or at least 0.25 wt %, or at least 0.5 wt % based on the weight ofthe debridement composition.

In some embodiments, the total amount of non-proteolytic enzyme in adebridement composition capable of debriding devitalized or necrotictissue can be at least 0.001 wt % to 60 wt % based on the total weightof the debridement composition. In some embodiments, the total amount ofnon-proteolytic enzyme can be at least 1 wt % to 50 wt %, or at least 2wt % to 40 wt %, or at least 5 wt % to 35 wt %, or at least 10 wt % to30 wt %. In some embodiments, the non-proteolytic enzyme is an amylase.In some embodiments, the non-proteolytic enzyme is α-amylase.

In some embodiments, the amount of non-proteolytic enzymatic debridementcomponent capable of debriding devitalized or necrotic tissue in thedebridement composition can be 100 wt %, or at least 99.5 wt %, or atleast 99 wt %, or at least 95 wt %, or at least 90 wt %, or at least 85wt %, or at least 80 wt %. In some embodiments, the amount ofnon-proteolytic enzymatic debridement component capable of debridingdevitalized or necrotic tissue in the debridement composition can be upto 100 wt %, or up to 99.5 wt %, or up to 99 wt %, or up to 95 wt %, orup to 90 wt %, or up to 85 wt %, or up to 80 wt %. In some embodiments,the amount of non-proteolytic enzymatic debridement component capable ofdebriding devitalized or necrotic tissue in the debridement compositioncan be at least 0.001 wt %, or at least 0.01 wt %, or at least 0.05 wt%, or at least 0.075 wt %, or at least 0.1 wt %, or at least 0.15 wt %.

In some embodiments, the amount of amylase in the debridementcomposition can be 100 wt %, at least 99.5 wt %, at least 99 wt %, atleast 95 wt %, at least 90 wt %, at least 85 wt %, or at least 80 wt %.In some embodiments, the amount of amylase in the non-proteolyticenzymatic debridement component can be 100 wt %, at least 99.5 wt %, atleast 99 wt %, at least 95 wt %, at least 90 wt %, at least 85 wt %, orat least 80 wt %, with the remainder of the non-proteolytic enzymaticdebridement component being other non-proteolytic enzymes. The amount ofα-amylase can be at least 10 wt %, at least 20 wt %, at least 30 wt %,at least 40 wt %, at least 50 wt %, at least 60 wt %, at least 70 t-%,at least 80 wt %, at least 90 wt %, or 100 wt % of the amylase content.In some embodiments, the remaining portion of the non-proteolyticenzymatic debridement component (20 wt % or less, 15 wt % or less, 10 wt% or less, 5 wt % or less, 1 wt % or less, 0.5 wt % or less) can be, butnot limited to, hydrolytic, lytic, and oxidative/reductive enzymesincluding, but are not limited to, hydrolytic, lytic, andoxidative/reductive enzymes selected from the group consisting oflipases, hyaluronidases, chondroitinases, heparanases, heparinases,peroxidases, xylanases, nucleases, phospholipases, esterases,phosphatases, isoamylases, maltases, glycosylases, galactosidases,cutinases, lactases, inulases, pectinases, mannanases, glucosidases,invertases, pectate lyases, reductases, oxidases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,glucanases, arabinosidases, sulfatases, cellulases, hemicellulases,laccases, mixtures thereof, and the like. Examples of proteolyticenzymes that can be present in the debridement composition include, butare not limited to, proteases and keratinases.

The debridement composition can include an aqueous media. In someembodiments, the aqueous media can have a pH in the range 3.0-10.0, orfrom 4.5-8.0, or from 5.5 to 7.5. in some embodiments, the debridementcomposition can have an osmolality of 10-340 mOsm/kg. Where thedebridement composition is an aqueous-based solution, gel or paste, awater-soluble polymer can be added to increase solution viscosity and toprolong residence time of the enzymatic composition on the necrotictissue, on the surface of a wound, or subcutaneously in a wound.

The enzymatic debridement composition can be applied to the devitalizedtissue as needed to dissolve necrotic debris in and around a wound. Forexample, in some embodiments the enzymatic debridement composition canbe in contact with the devitalized tissue for about 1 to 48 hours, 1 to24 hours, 1 to 12 hours, 1 to 8 hours, 1 to 4 hours, or 1 to 2 hoursbefore removal.

In some embodiments, the amylase formulation can be removed from a woundby wiping or by rinsing with saline or water. These steps may berepeated as needed. A wide variety of wounds can be treated with thedebridement compositions described herein, including full and partialthickness burn wounds, diabetic ulcers, ulcerative lesions, principallypressure (decubitus) ulcers, venous ulcers, trophic ulcers, surgicalwounds such as amputation, incisional, traumatic and pyogenic wounds,infected wounds by microorganisms, donor and receptor skin graft wounds,malignancy, cysts, radiation wounds, sunburn, and frostbite.

In some embodiments, the enzymatic debridement composition can beinjected into the devitalized tissue. A penetration enhancer may also beutilized to enhance transdermal delivery of solutions, gels, creams,lotions, aerosols, and sprays. Penetration enhancers that can be used inthe debridement compositions described herein include, but are notlimited to, fatty acids such as branched and linear C₆-C₁₈ saturatedacids, unsaturated acids, such as C₁₄ to C₂₂, oleic acid,cis-9-octadecenoic acid, linoleic acid, linolenic acid, fatty alcohols,such as saturated C₈-C₁₈ terpenes, such as d-limonene, α-pinene,3-carene, menthone, fenchone, pulegone, piperitone, eucalyptol,chenopodium oil, carvone, menthol, α-terpineol, terpinen-4-ol, carveol,limonene oxide, pinene oxide, cyclopentane oxide, triacetin, cyclohexaneoxide, ascaridole, 7-oxabicylco[2,2,1]heptane, 1,8-cineole, glycerolmonoethers, glycerol monolaurate, glycerol monooleate, isostearylisostearate, isopropyl myristate, isopropyl palmitate, isopropyllanolate, pyrrolidones, such as N-methyl-2-pyrrolidone,1-ethyl-2-pyrrolidone, 5-methyl-2-pyrrolidone,1,5-dimethyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid,N-hexyl-2-pyrrolidone, N-lauryl-2-pyrrolidone,1-dodecylazacycloheptan-2-one, 4-decyloxazolidin-2-one,N-dodecylcaprolactam, and 1-methyl-3-dodecyl-2-pyrrolidoneN-n-butyl-N-n-dodecylacetamide, N,N-di-n-dodecylacetamide,N-cycloheptyl-N-n-dodecylacetamide and N,N-di-n-propyldodecanamide,urea, 1-dodecylurea, 1,3-didodecylurea, 1,3-diphenyl urea, dimethylsulfoxide, decylmethyl sulfoxide, tetradecylmethyl sulfoxide,cyclodextrins, and combinations thereof. Also effective penetrationenhancers include 1-alkyl-2-piperidinones, 1-alkyl-2-azacycloheptanones,such as 1-dodecyazacycloheptan-2-one, 1,2,3-alkanetriols, such as1,2,3-nonanetriol, 1,2-alkanediols, n-,2-(1-alkyl)-2-methyl-1,3-dioxolanes, oxazolidinones, such as4-decyloxazolidin-2-one, N,N-dimethylalkanamides, 1,2-dihydroxypropylalkanoates, such as 1,2-dihydroxypropyl decanoate, 1,2-dihydroxypropyloctanoate, sodium deoxycholate, trans-3-alken-1-ols, cis-3-alken-1-ols,and trans-hydroxyproline-N-alkanamide-C-ethylamide, and combinationsthereof. In some embodiments, the penetration enhancers can includehydrophobic esters isopropyl myristate, isopropyl palmitate, orcombinations thereof.

Because of the possibly of infection in devitalized necrotic tissue, thedebridement composition can include a biological agent in an amountsufficient to hinder or eradicate microorganisms. Such biological agentsinclude, but are not limited to, antibiotics, antiseptics,anti-infective agents, antimicrobial agents, antibacterial agents,antifungal agents, antiviral agents, antiprotozoal agents, sporicidalagents, antiparasitic agents. In some embodiments, the biological agentis biodegradable and non-cytotoxic to human and animal cells. Usefulbiocidal agents include, but are not limited to, biguanides, such aspoly(hexamethylene biguanide hydrochloride) (PHMB), a low molecularweight synthetic polycation, chlorhexidine and its salts, such aschlorhexidine digluconate (CHG), and alexidine and its salts, where thelatter two are bis(biguanides).

In some embodiments, the biguanide is PHMB because of its high biocidalactivity against microorganisms, combined with its biodegradation andlow cytotoxicity. PHMB is primarily active against Gram negative andGram positive bacteria, fungi, and viruses, and also serves as apreservative. In contrast to antibiotics, which are considered regulatedpharmaceutical drugs, to which bacterial resistance can occur, suchresistance does not occur with PHMB. In general, an antimicrobial agentis defined as a substance that kills microorganisms or inhibits theirgrowth or replication, while an anti-infective agent is defined as asubstance that counteracts infection by killing infectious agents, suchas microorganisms, or preventing them from spreading. Often, the twoterms are used interchangeably. As used herein, PHMB is considered anantimicrobial agent.

In some embodiments, the debridement composition is an aqueousdebridement composition. In some embodiments, aqueous debridementcompositions described herein can include biocidal PHMB at aconcentration ranging from 0.01 wt % (100 ppm) to 1 weight % (10,000ppm), or ranging from 0.05 wt % (500 ppm) to 0.5 wt % (5,000 ppm), orranging from 0.1 wt % (1,000 ppm) to 0.15 wt % (1,500 ppm) based on thetotal weight of the debridement composition. Bis(biguanides), such asalexidine and its salts and chlorhexidine and its salts, can also beadded to the antimicrobial compositions in concentrations from 0.001 wt% (10 ppm) to 4.0 wt % (40,000 ppm).

The interaction of low molecular weight PHMB polycation (molecularweight 2,400 Daltons) with amylase, a negatively charged, high molecularweight amylase (molecular weight 55,000 Daltons) at physiological pH cangenerate a protein-polyelectrolyte complex of PHMB ionically interactedwith amylase. In a wound, PHMB may be released from theprotein-polyelectrolyte complex in a continuous release fashion as afunction of the amount of low molecular weight cation in bodily fluids(such as sodium, potassium, calcium and magnesium ions) displacing thecationic PHMB ionic interaction with anionic amylase sites.

The dosage at which the therapeutic amylase compositions areadministered is dependent upon the source of the amylase, the activity(i.e., the number of Units involved), the size of necrotic tissue, theage of the patient, the availability of clinical care, and the incidenceof infection. The amount of therapeutic amylase that may be administeredup to twice per day can range from application of a powder (at 100 wt %)to a dilute solution (of about 0.001 wt %). In some embodiments, theactivity of the amylase can range from 250 Units to 250,000 Units pergram of enzyme in 1 gram of the debridement composition.

The debriding composition on necrotic tissue can be performed by aclinician, such that the patient is not in any further medicaldifficulty. The amylase method of debridement can be performed incombination with other known debriding methods.

As used herein, “debridement” has its standard meaning and includes theremoval of lacerated, devitalized, dead, damaged, infected orcontaminated tissue, foreign bodies, and other debris from the wound bedin order to expose healthy tissue. Nonviable or necrotic tissue caneither be eschar or slough.

As used herein, “necrotic tissue” has its standard meaning and includesdead tissue that contains dead cells and debris, as a consequence of thefragmentation of dying cells. The color of necrotic tissue changes fromred to brown, or black or purple, as it becomes more dehydrated, finallyresulting in a black, dry, thick, and leathery eschar structure, whichcan occur in a wide variety of wound types, including burns and alltypes of chronic wounds.

As used herein, “tissue” in the body includes muscle tissue, connectivetissue, epithelial tissue, and nervous tissue.

As used herein, “eschar” has its standard meaning and includes forms ofnecrotic tissue. It is the leather-like covering on the wound at theskin surface, and it can be either hard or pliable.

As used herein, “slough” has its standard meaning and includes moistnecrotic tissue. This type of devitalized tissue is soft, moist andoften stringy in consistency and is usually yellow, white or grey incolor.

As used herein, “devitalized tissue” has its standard meaning andincludes tissue devoid of vitality or life, that is, dead tissue.

As used herein, “proteolytic enzyme” has its standard meaning andincludes enzymes that independently cleave (digest, break, hydrolyze)the long chainlike polymer molecules of proteins into shorter fragmentsof peptides and, eventually, into their basic components of amino acids.

As used herein, “excise” has its standard meaning and includes asurgical procedure requiring incision utilizing a scalpel or other sharpinstrument through the deep dermis (including subcutaneous and deepertissues).

As used herein, a covalent bond that is formed between a carbohydratemolecule and another molecule, particularly between two monosaccharidemoieties, is a “glycosidic bond” or “glycodisic linkage.”

As used herein, “α-1,4-glycosidic linkages” are bonds that are normallyformed between the carbon-1 on one sugar and the carbon-4 on anothersugar moiety in a polysaccharide. An α-glycosidic bond is formed whenthe —OH group on carbon-1 is below the plane of the glucose ring. On theother hand, a β-glycosidic bond is formed when it is above the plane.For example, cellulose is formed of glucose molecules linked by 1-4β-glycosidic bonds, whereas starch is composed of 1-4 α-glycosidicbonds.

As used herein, “α-amylase” includes naturally occurring α-amylases aswell as recombinant α-amylases, wherein recombinant α-amylase means anα-amylase in which the DNA genetic sequence encoding the naturallyoccurring α-amylase is modified to produce a mutant DNA sequence thatencodes the substitution, insertion or deletion of one or more aminoacids in the α-amylase sequence compared to the naturally occurringα-amylase.

As used herein, “amylolytic” is characterized by or capable of theenzymatic digestion of starch into dextrins and sugars, particularly byamylase.

As used herein, the amount of enzyme utilized is expressed in weightpercent and its activity is given in Units of activity per gram, where a“Unit” is defined as the amount of enzyme that catalyzes the conversionof 1 micromole of substrate per minute.

As used herein, “surfactant” has its standard meaning and includescompounds that lower the surface tension (or interfacial tension)between two liquids or between a liquid and a solid and includesemulsifying agents, emulsifiers, detergents, wetting agents, andsurface-active agents.

As used herein, “microemulsion” has its standard meaning and includesthermodynamically stable mixtures of oil, water (and/or hydrophiliccompound) and surfactant. Microemulsions include three basic types:direct (oil dispersed in water, o/w), reverse (water dispersed in oil,w/o) and bicontinuous. Microemulsions are optically clear because thedispersed micelles have a diameter that is less than the wavelength ofvisible light (e.g., less than 380 nanometers, less than 200 nanometers,or less than 100 nanometers) in diameter. In the absence of opacifiers,microemulsions are optically clear, isotropic liquids.

As used herein, “reverse microemulsion” has its standard meaning andincludes a microemulsion comprising a hydrophilic phase suspended in acontinuous oil phase. A reverse microemulsion can include droplets of ahydrophilic phase (e.g., water, alcohol, or a mixture of both)stabilized in an oil phase by a reverse emulsion surfactant. In suchinstances, a hydrophilic active agent can be solubilized in thedroplets. However, in other instances, the reverse microemulsion can befree of water and/or alcohol, and the hydrophilic active agent can bedirectly solubilized in the oil phase by the reverse emulsionsurfactant.

As used herein, “hydrophilic” has its standard meaning and includescompounds that have an affinity to water and can be ionic or neutral orhave polar groups in their structure that attract water. For example,hydrophilic compounds can be miscible, swellable or soluble in water.

As used herein, “aqueous” compositions refer to a spectrum ofwater-based solutions including, but not limited to, homogeneoussolutions in water with solubilized components, emulsified solutions inwater stabilized by surfactants or hydrophilic polymers, and viscous orgelled homogeneous or emulsified solutions in water.

As used herein, “solid” compositions refer to dried formulations inpowder or film form.

As used herein, an enzyme is “soluble” or “solubilized” if the amount ofenzyme present in the solvent system is dissolved in the solvent systemwithout the enzyme forming a precipitate or visible, swollen gelparticles in solution.

As used herein, “non-stinging” means that the formulation does not causea sharp, irritatingly, burning or smarting pain as a result of contactwith a biological surface.

As used herein, “volatile” has its standard meaning, that is, it canevaporate rapidly at normal temperatures and pressures. For example, asolvent is volatile if one drop (0.05 mL) of the solvent will evaporatecompletely between 20-25° C. within 5 minutes, or within 4 minutes, orwithin 3 minutes, or within 2 minutes, or within 1 minute, or within 30seconds, or within 15 seconds.

As used herein, an “antimicrobial agent” is defined as a substance thatkills microorganisms or inhibits their growth or replication, while ananti-infective agent is defined as a substance that counteractsinfection by killing infectious agents, such as microorganisms, orpreventing them from spreading. Often, the two terms are usedinterchangeably. Antibiotics are considered those substances that wereoriginally produced by a microorganism or synthesized with activeproperties that can kill or prevent the growth of another microorganism.The term antibiotic is commonly used to refer to almost any prescribeddrug that attempts to eliminate infection. Antimicrobial agents do notcause biocidal resistance such as can occur with antibiotics, whereinantibiotic resistance to a drug can occur. Antimicrobial agents have abroad spectrum of activity against bacteria, fungi, viruses, protozoaand prions. Examples of antimicrobial agents include biguanides, such aspoly(hexamethylene biguanide hydrochloride) (PHMB), chlorhexidine andits salts, alexidine and its salts, povidone/iodine, cadexomer iodine,silver sulfadiazine, nanocrystalline silver, ionic silver, honey, dilutebleaching agents such as sodium hypochlorite and hypochlorous acid,hydrogen peroxide, organic peroxides such as benzoyl peroxide, alcoholssuch as ethanol and isopropanol, anilides such as triclocarban,bisphenols such as triclosan, chlorine compounds such as chlorinedioxide and N-chloramines, and quaternary ammonium compounds such asbenzalkonium chloride, benzethonium chloride, cetyltrimethylammoniumchloride, cetylpyridinium chloride, and alkyltrimethylammonium bromides,as well as miconazole, clotrimazole, ketoconazole, fluconazole, crystalviolet, amphotericin B, tee tree oil, and the like. Biguanides, such asPHMB, are useful in the debridement compositions described herein.

A polymeric biguanide useful in the debridement compositions describedherein is poly(hexamethylene biguanide), commercially available fromArch Chemicals, Inc., Smyrna, Ga. under the trademark Cosmocil™ CQ.Poly(hexamethylene biguanide) polymers are also referred to aspoly(hexamethylene biguanide) (PHMB), poly(hexamethylene bisbiguanide)(PHMB), poly(hexamethylene guanide) (PHMB), poly(aminopropyl biguanide)(PAPB), poly[aminopropyl bis(biguanide)] (PAPB), polyhexanide andpoly(iminoimidocarbonyl)iminohexamethylene hydrochloride; however, PHMBis the preferred abbreviation. PHMB is a broad spectrum antimicrobialand has been used in contact lens multipurpose solutions, wound rinsingsolutions, wound dressings, perioperative cleansing products,mouthwashes, surface disinfectants, food disinfectants, veterinaryapplications, cosmetic preservatives, paper preservatives, secondary oilrecovery disinfectants, industrial water treatments, and in swimmingpool cleaners. It is normally obtained commercially in the hydrochlorideform in water. Other antimicrobial polymers can also be added, such aspolyquaternium 1, polyquaternium 6, polyquaternium 10, cationic guar,and water-soluble derivatives of chitosan.

As used herein, an “antibiotic” is a medicine or drug, usuallyprescribed, such as penicillin, streptomycin, chloramphenicol, andtetracycline, usually produced by various microorganisms, that inhibitsthe growth of or destroys microorganisms, used primarily in thetreatment of infectious disease. Antibiotics are considered thosesubstances that were originally produced by a microorganism orsynthesized with related active properties that can kill or prevent thegrowth of another microorganism. The term antibiotic is commonly used torefer to almost any prescribed drug that attempts to eliminateinfection.

As used herein, “antibiotic resistance” is the ability of bacteria andother microorganisms to resist the effects of an antibiotic to whichthey were once susceptible.

The debridement composition described herein can include a biocidalmonoalkyl glycol, glycerol alkyl ether, and monoacyl glycerol at acombined concentration of from 0.05 wt % (500 ppm) to 4 wt % (4,000ppm), or from 0.1 wt % (1,000 ppm) to 1 wt % (10,000 ppm), or from 0.4wt % (4,000 ppm) to 0.6 wt % (6,000 ppm) based on the weight of thedebridement composition. The monoalkyl glycol, glycerol alkyl ether, andmonoacyl glycerol can be hydrophobic.

Examples of monoalkyl glycols useful in the debridement compositionsdescribed herein include, but are not limited to, 1,2-propanediol(propylene glycol), 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol,1,2-heptanediol, 1,2-octanediol (caprylyl glycol), 1,2-nonanediol,1,2-decanediol, 1,2-undecanediol, 1,2-dodecanediol, 1,2-tridecanediol,1,2-tetradecanediol, 1,2-pentadecanediol, 1,2-hexadecanediol,1,2-heptadecanediol and 1,2-octadecanediol. Non-vicinal glycols can alsobe added to enhance biocidal activity. Exemplary, non-vicinal glycolsinclude, but are not limited to, 2-methyl-2,4-pentanediol,1,3-butanediol, diethylene glycol, triethylene glycol, and glycolbis(hydroxyethyl) ether.

Examples of glycerol alkyl ethers useful in the debridement compositionsdescribed herein include, but are not limited to, 1-O-heptylglycerol,1-O-octylglycerol, 1-O-nonylglycerol, 1-O-decylglycerol,1-O-undecylglycerol, 1-O-dodecylglycerol, 1-O-tridecylglycerol,1-O-tetradecylglycerol, 1-O-pentadecylglycerol, 1-O-hexadecylglycerol(chimyl alcohol), 1-O-heptadecylglycerol, 1-O-octadecylglycerol (batylalcohol), 1-O-octadec-9-enyl glycerol (selachyl alcohol), glycerol1-(2-ethylhexyl) ether (also known as octoxyglycerin, 2-ethylhexylglycerin, 3-(2-ethylhexyloxy)propane-1,2-diol, and Sensiva® SC 50),glycerol 1-heptyl ether, glycerol 1-octyl ether, glycerol 1-decyl ether,and glycerol 1-dodecyl ether, glycerol 1-tridecyl ether, glycerol1-tetradecyl ether, glycerol 1-pentadecyl ether, glycerol 1-hexadecylether and glycerol 1-octadecyl ether.

Examples of monoacyl glycerols useful in the debridement compositionsdescribed herein include, but are not limited to, 1-O-decanoylglycerol(monocaprin), 1-O-undecanoylglycerol, 1-O-undecenoylglycerol,1-O-dodecanoylglycerol (monolaurin, also called glycerol monolaurate andLauricidin®), 1-O-tridecanoylglycerol, 1-O-tetradecanoylglycerol(monomyristin), 1-O-pentadecanoylglycerol, 1-O-hexadecanoylglycerol,1-O-heptadecanoylglycerol, and 1-O-octanoylglycerol (monocaprylin). Ingeneral, glycerols substituted in the 1-O-position are more preferredthan those substituted in the 2-O-position, or disubstituted in the 1-Oand 2-O positions.

As used herein, “hydrophobic” refers to repelling water, being insolubleor relatively insoluble in water, and lacking an affinity for water.Hydrophobic compounds with hydrophilic substituents, such as vicinaldiols, may form emulsions in water, with or without added surfactant.

As used herein, “amphoteric” refers to a mixture of cationic and anioniccharges on a molecule or polymer in which overall charge is locally pHdependent, whereas “ampholytic” has an equal number of cationic andanionic charges over a broad pH range.

As used herein, an “excipient” is a usually inert substance that forms avehicle, such as a liquid, fluid, or gel, that solubilizes or dispersesan enzyme or other added ingredients. The amylase compositions caninclude one or more additional surfactants to enhance necrotic tissueremoval. Suitable surfactants include, but are not limited to, cationic,anionic, nonionic, amphoteric and ampholytic surfactants. In someembodiments, the surfactants are nonionic and amphoteric surfactants. Insome embodiments, the surfactant can be present in an amount rangingfrom 0% to 10 wt % based on the weight of the debridement composition.In some embodiments, the surfactant can be present in an amount of atleast 0.01 wt %, or at least 0.1 wt %, or at least 0.25 wt %, or atleast 0.5 wt %, or at least 1 wt %, based on the weight of thedebridement composition. The surfactants can have an HLB(hydrophilic-lipophilic balance) value of 18-30 in order to maintain theenzymes' catalytic structure in solution as well as not hindering thebiocidal activity of any added antimicrobial agents, while facilitatinga non-cytotoxic solution. The high values of the HLB representsurfactants that are more hydrophilic than those with lower HLB values.

Suitable nonionic surfactants include, but are not limited to, theethylene oxide/propylene oxide block copolymers of poloxamers, reversepoloxamers, poloxamines, and reverse poloxamines. Poloxamers andpoloxamines are preferred, and poloxamers are most preferred. Poloxamersand poloxamines are available from BASF Corp. under the respective tradenames of Pluronic® and Tetronic®. Suitable Pluronic surfactants comprisebut are not limited to Pluronic F38 having a HLB of 31, Pluronic F68having a HLB of 29, Pluronic 68LF having a HLB of 26, Pluronic F77having a HLB of 25, Pluronic F87 having a HLB of 24, Pluronic F88 havinga HLB of 28, Pluronic F98 having a HLB of 28, Pluronic F108 having a HLBof 27, Pluronic F127 (also known as Poloxamer 407) having a HLB of18-23, and Pluronic L35 having a HLB of 19. An exemplary poloxaminesurfactant of this type is Tetronic 1107 (also known as Poloxamine 1107)having an HLB of 24.

In addition to the above, other neutral surfactants may be added, suchas for example polyethylene glycol esters of fatty acids, e.g., coconut,polysorbate, polyoxyethylene or polyoxypropylene ethers of higheralkanes (C₁₂-C₁₈), polysorbate 20 available under the trademark Tween20, polyoxyethylene (23) lauryl ether available under the trademark Brij35, polyoxyethylene (40) stearate available under the trademark Myrj 52,and polyoxyethylene (25) propylene glycol stearate available under thetrademark Atlas G 2612, all available by Akzo Nobel, Chicago, Ill. Otherneutral surfactants include nonylphenol ethoxylates such as nonylphenolethoxylates, Triton X-100, Brij surfactants of polyoxyethylenevegetable-based fatty ethers, Tween 80, decyl glucoside, and laurylglucoside.

Amphoteric surfactants suitable for use in antimicrobial compositionsaccording to the present invention include materials of the type offeredcommercially under the trademark Miranol (Rhodia). Another useful classof amphoteric surfactants is exemplified by betaines, includingcocoamidopropyl betaine, undecylenamidoalkylbetaine, andlauramidoalkylbetaine and sodium cocoamphoacetate. Amphotericsurfactants are very mild and have excellent dermatological properties,making them particularly suited for use in personal care applications.

The debridement composition may further comprise a chelating agent at aconcentration of from 0.01 weight % to 1 weight %. For example, thechelating agent can be present in an amount of at least 0.01 wt %, or atleast 0.03 wt %, or at least 0.05 wt %, or at least 0.1 wt %, or atleast 0.50 wt %, or at least 0.75 wt %, or at least 1.0 wt %. Thechelating agent can be selected from the group that includes, but is notlimited to, ethylenediaminetetraacetic acid (EDTA), nitrilotriaceticacid, nitrilotripropionic acid, diethylenetriaminepentaacetic acid,2-hydroxyethylethylenediaminetriacetic acid,1,6-diaminohexamethylenetetraacetic acid,1,2-diaminocyclohexanetetraacetic acid,O,O′-bis(2-aminoethyl)ethyleneglycoltetraacetic acid,1,3-diaminopropanetetraacetic acid,N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid,ethylenediamine-N,N′-diacetic acid, ethylenediamine-N,N′-dipropionicacid, triethylenetetraaminehexaacetic acid,ethylenediamine-N,N′-bis(methylenephosphonic acid), iminodiacetic acid,monosodium-N-lauryl-β-iminodipropionic acid (sodiumlauriminodipropionate, Deriphat® 160C), N, N-bis(2-hydroxyethyl)glycine,1,3-diamino-2-hydroxypropanetetraacetic acid,1,2-diaminopropanetetraacetic acid,ethylenediaminetetrakis(methylenephosphonic acid),N-(2-hydroxyethyl)iminodiacetic acid, biphosphonates, editronate, andsalts thereof.

The debridement compositions can also contain chlorophyllin and itswater-soluble derivatives in order to reduce local inflammation, promotehealing, and control odor. It has been reported that in treating woundsthat require debridement, some of the end products of proteolysis aremucoproteins, which often produce an irritating action and otherdeleterious actions on the tissue in contact with said end products. Inorder to control such undesirable effects, the incorporation of awater-soluble chlorophyll derivative has been incorporated into aproteolytic debridement formulation, generally in an amount of 0.05 to1% by weight of the total composition, and preferably in an amount of0.1% to 0.5% (U.S. Pat. No. 2,917,433). The water-soluble chlorophyllswhich may be used for the aforementioned purpose are exemplified bythose disclosed in U.S. Pat. No. 2,120,667, including especially sodiumor potassium copper chlorophyllin, sodium or potassium magnesiumchlorophyllin and sodium or potassium iron chlorophyllin. The preferredwater-soluble chlorophyll for this purpose was a mixture of sodium andpotassium copper chlorophyllin, predominantly the potassium salt.

Other applications of chlorphyllin in wound treatment are exemplified byInternational Patent Publication Number WO 2008/063229, wherein aprotease enzyme debridement composition is a hydrophilic ointment thatadditionally comprises a supplemental agent that minimizes damage tohealthy normal cells from compounds released by dying cells, wherein theagent is optionally sodium copper chlorophyllin, wherein the range ofthe stoichiometric ratio of moles of chlorophyllin administered to molesof debridement enzyme administered is optionally 0.1 to 10.0, 0.3 to3.0, or 1.0.

Sodium copper chlorophyllin is also a food coloring agent and is knownas natural green 3. Its addition to a debridement product also addscoloration, which is helpful in visualizing the removal of thedebridement composition.

If pain reduction is required during debridement, the formulation canfurther comprise analgesic agents, anesthetic agents, and neuropathicpain agents, such as lidocaine, capsaicin, benzocaine, tetracaine,prilocaine, bupivacaine, levobupivacaine, procaine, carbocaine,etidocaine, mepivacaine, nortripylene, amitriptyline, pregabalin,diclofenac, fentanyl, gabapentin, non-steroidal anti-inflammatoryagents, salicylates, combinations thereof, and the like.

Whereas α-amylases catalyze the hydrolysis of internal α-(1-4)-linkagesof glucose polymers as their main reaction, some α-amylases,particularly saccharifying amylases, however, catalyze transferreactions in addition to hydrolysis (U.S. Pat. No. 8,486,664;International Patent Application Number WO 2012/013646). Theseα-amylases are capable of transferring glycoside residues to lowmolecular weight alcohols as well as to water, a property related to thetransferase activity of the glycosidases. It is not known whether such atransglycosylation process is operative in the debridement of necrotictissue as related to this invention.

In some embodiments, a kit that includes a container containingdebridement composition according to any of the variations describedherein, and instructions for using the debridement composition fordebridement of devitalized tissue is described. The instructions caninclude contacting the debridement composition with an area of skin inneed of debridement. The instructions can include repeating thecontacting step at regular intervals. The regular intervals can be atleast once a day, or at least twice a day (every 12 hours), or at leastthree times a day (every 8 hours). The instructions can include mixingand or diluting the debridement composition in a solvent or othercarrier liquid. The instructions can include removal of the necrotictissue treated debridement composition by wiping and by solvent rinsing.

A method of debridement of devitalized tissue is also described. Themethod can include contacting a debridement composition according to anyof the variations described herein with an area of skin in need ofdebridement, such as by a powder, liquid, gel, hydrogel, foam, paste,spray, or film. In some embodiments, the debridement composition isapplied to a wound dressing, such as gauze, cloth, fiber, alginate,hydrocolloid, composite, or film. In some embodiments, the wounddressing is composed of natural or synthetic components, or combinationsthereof. The method can also include abrading and/or removing thedebridement composition after a given period of time. The method caninclude repeating the contacting step at regular intervals. In someembodiments, the regular intervals can be at least once a day, or atleast twice a day (every 12 hours), or at least three times a day (every8 hours). In some embodiments, the method also includes removingdebrided tissue from the area of skin.

It is an object of the compositions, kits, and methods described hereinto provide for enzymatic debridement of necrotic debris and eschar.

It is an object of the compositions, kits, and methods described hereinto provide for enzymatic debridement treatment of chronic wounds, acutewounds and burn wounds.

It is a further object of the compositions, kits, and methods describedherein to provide enzymatic debridement of necrotic tissue not basedpredominantly on peptide cleavage/hydrolysis by proteases.

It is a further object of the compositions, kits, and methods describedherein to provide enzymes for debridement that cleave carbohydrateα-1,4-glycodisic linkages in polysaccharides or glycosidic linkages withproteins.

It is a further object of the compositions, kits, and methods describedherein to provide carbohydrate hydrolytic enzymes for enzymaticdebridement based on the amylase family of enzymes.

It is a further object of the compositions, kits, and methods describedherein to treat necrotic wounds comprising the step of administering aneffective amount of an amylase enzyme.

It is a further object of the compositions, kits, and methods describedherein to provide carbohydrate hydrolytic enzymes for enzymaticdebridement based upon α-amylase.

It is a further object of the compositions, kits, and methods describedherein to provide carbohydrate hydrolytic enzymes for enzymaticdebridement based upon β-amylase.

It is a further object of the compositions, kits, and methods describedherein to provide carbohydrate hydrolytic enzymes for enzymaticdebridement based upon γ-amylase in combination with α-amylase and withβ-amylase.

It is a further object of the compositions, kits, and methods describedherein to provide carbohydrate hydrolytic enzymes for enzymaticdebridement based upon the amylase family, selected from combinations ofα-amylase, with β-amylase, and γ-amylase.

It is a further object of the compositions, kits, and methods describedherein to provide for enzymatic tissue debridement based predominantlyon α-amylase, with a minimum (20 wt % or less) of other families ofenzymes.

It is a further object of the compositions, kits, and methods describedherein to provide for enzymatic tissue debridement wherein families ofhydrolytic cleavage enzymes other than amylases include 20 wt % or lessof proteases, chondroitinases, hyaluronidases, lipases, glycosidases,heparanases, dermatanases, pullulanases, N-acetylglucosaminidase,lactases, phospholipases, transglycosylases, esterases, thioesterhydrolyases, sulfatases, escharases, nucleases, phosphatases,phosphodiesterases, mannanases, mannosidases, isoamylases, lyases,inulinases, keratinases, tannases, pentosanases, glucanases,arabinosidases, pectinases, cellulases, chitinases, xylanases,cutinases, pectate lyases, hemicellulases, combinations thereof, and thelike.

It is a further object of the compositions, kits, and methods describedherein to provide for enzymatic tissue debridement wherein families ofenzymes other than amylases include 20 wt % or less of oxidases,peroxidases, glucose oxidases, catalases, oxidoreductases,phenoloxidases, laccases, lipoxygenases, isomerases, ligninases,combinations thereof, and the like.

It is a further object of the compositions, kits, and methods describedherein to provide for debridement based upon α-amylase, wherein theα-amylase is administered as a powder, gel, paste, liquid, ointment,foam, bandage, mesh or dressing.

It is a further object of the compositions, kits, and methods describedherein to provide for debridement based upon α-amylase that isadministered topically or subcutaneously.

It is a further object of the compositions, kits, and methods describedherein to provide for debridement based upon α-amylase, wherein theα-amylase is applied in a hydrophilic or aqueous medium.

It is a further object of the compositions, kits, and methods describedherein to provide a pleasing fragrance to the debridement compositions.

It is a further object of the compositions, kits, and methods describedherein to apply chlorophyllin to a wound to reduce local inflammation,promote healing, and control odor.

It is a further object of the compositions, kits, and methods describedherein to provide a wound dressing for amylase treatment comprised ofgauze, mesh, cloth, fiber, foam or film.

It is a further object of the compositions, kits, and methods describedherein to provide for preserved formulations of amylase.

It is a further object of the compositions, kits, and methods describedherein to provide for antimicrobial formulations of amylase.

It is a further object of the compositions, kits, and methods describedherein to provide for antimicrobial formulations of amylase that reduceor eliminate Gram positive and Gram negative bacteria in wounds andbiological surfaces.

It is a further object of the compositions, kits, and methods describedherein to provide for antimicrobial formulations of amylase that reduceor eliminate yeast in wounds and biological surfaces.

It is a further object of the compositions, kits, and methods describedherein to provide for antimicrobial amylase formulations thatincorporate antimicrobial essential oils to augment the antimicrobialactivity of the compositions.

It is a further object of the compositions, kits, and methods describedherein to provide for debridement based upon α-amylase by a surfactantcapable of solubilizing, swelling, or hydrating devitalized tissue oreschar.

It is a further object of the compositions, kits, and methods describedherein to provide for exfoliation based upon α-amylase with a surfactantcapable of solubilizing, swelling, or hydrating devitalized skin.

It is a further object of the compositions, kits, and methods describedherein to provide for α-amylase-based debridement a hydrophilic polymercapable of increasing viscosity or causing gelation of the formulation.

It is a further object of the compositions, kits, and methods describedherein to provide amylase in kit form for treatment of wounds, necrotictissue, and devitalized tissue.

Examples

The following ingredients and their abbreviations are used in thisinvention:

Enzymes

α-Amylase #1, porcine pancreas, 30 U/mg, Sigma Aldrich, A3176-500KU, lotSLBF3831V.α-Amylase #2, porcine pancreas, (contains 0.2% protease), 230 U/mg, LeeBioSolutions, lot M60404.α-Amylase #3, porcine pancreas, (contains 0.05% protease), 210 U/mg, LeeBioSolutions, lot P70442.α-Amylase #4, human saliva, 117.5 U/mg, Sigma Aldrich, lot SLBB8953V.α-Amylase #5, Bacillus licheniformis, 500-1500 U/mg, Sigma Aldrich, lotSLBG8595V.α-Amylase #6, Bacillus subtilis spp., powder, 7278 U/mg, DyadicInternational, lot ADY4001.α-Amylase #7, Bacillus subtilis spp., solution, 1269 U/mg, DyadicInternational, lot ASP3001.α-Amylase #8a, Aspergillus oryzae, powder, Enzyme DevelopmentCorporation, sample S27837.α-Amylase #8b, Aspergillus oryzae, powder, Enzyme DevelopmentCorporation, sample S28568.1-Amylase, barley, 41.6 U/mg, Sigma Aldrich, lot SLBC2932V.γ-Amylase #1, Aspergillus niger, 129.2 U/mg, Sigma Aldrich, lotBCBD1453V.

γ-Amylase #2, Rhizopus spp., MyBiosource Inc., lot 22200303.

Bromelain, pineapple stem, 3-7 U/mg, Sigma Aldrich, lot SLBG2202VCollagenase, Type I, Clostridium histolyticum, 125 U/mg, Sigma Aldrich,C0130-100UG, lot SLBH5757V.Lipase #1, porcine pancreas, 30-90 U/mg, Sigma Aldrich, lot SLBH6427V.Lipase #2, porcine pancreas, (contains <0.05% protease), 360 U/mg, LeeBioSolutions, 400-10, lot R24160.

Other Ingredients AC, Antimicrobial Composition, Water, 95.5 wt %, PHMB0.1 wt %, EDTA 0.065 wt %, P407 2 wt %, HPMC, 2 wt %, SC50, 0.3 wt %,SC10, 0.1 wt %, pH 5.5.

CHG, Chlorhexidine gluconate, Spectrum Chemicals, lot ZQ1023.Collagen, type I, rat tail, Corning Inc., 354236, lot 3298599.

DC 193, PEG-12 Dimethicone, Dow Corning, lot 0002250697 Dulbecco'sPhosphate Buffered Saline, DPBS, pH 7.1, Sigma Aldrich, D8537, lotRNBC1143.

EDTA, Ethylenediaminetetraacetic acid di-, tri-sodium salts, SpectrumChemicals, lots 1AE0430, YL0044.

Glycerin, Quality Choice, lot 519675. Hydroxypropylmethylcellulose(HPMC), Amerchol Corp., lot WF15012N01. Mineral Oil, CVS, lot 5BF0201.

PEG 400, Poly(ethylene glycol), 400 M_(n), Sigma-Aldrich, lot MKBD2642V.

Petrolatum, Vaseline, lot 02011HU00.

PHMB, Poly(hexamethylene biguanide hydrochloride), Cosmocil™ CQ, ArchChemical, lot 11RC116995.PHMB, Poly(hexamethylene biguanide hydrochloride), Cosmocil™ CQ, ArchPersonal Care, lot 137261.

P407, Poloxamer 407, Pluronic F127, Spectrum Chemicals, lot 1AD0265.

P40S, Polyoxyl 40 stearate, NF, Spectrum, lot 1DH0918.

Polymer JR-30M, Amerchol, lot XL2850GRXA.

Propylene glycol, USP, Spectrum, lot 1 EB0394.SA, Stearyl alcohol, NF, Spectrum, lot 2CL0259.

SC10, Sensiva® SC 10, 1,2-Dihydroxyoctane), Schulke & Mayr, lot 1178933.

SC50, Sensiva® SC 50, Glycerol 1-(2-ethylhexyl) ether), Schulke & Mayr,lot 1179743.SM, Sorbitan monostearate, NF, Spectrum, lot 1DF0797

Sodium Hydroxide, Puritan 50% NaOH, UN1824, lot 011043. Urea, SigmaAldrich, lot SLBF4607V.

Water, Deionized, adjusted to pH 7.WPJ, White petroleum jelly, USP, Spectrum, lot 1CK0612.

Collagen Gel Digestion

In order to determine if an α-amylase contained a protease and thus wasable to cleave a collagen gel (i.e., a protein-based gel), collagen geldigestion was studied by rheology under varied frequency conditionsusing α-amylase and collagenase as potential digesting enzymes. Ifα-amylase had no digestion of the collagen gel, its debridement activityof tissue would not be based upon any contamination by a protease, andthus not by hydrolysis of peptide bonds of collagen polymer chains.

Collagen gels were prepared at 2.0 mg/mL using collagen type I. Gelswere prepared by mixing 500 μL collagen (˜4.1 mg/mL), 500 μL Dulbecco'sphosphate buffered saline (DPBS), and 10 μL 1 N NaOH (diluted fromPuritan 50% NaOH). Solid collagen hydrogels were formed after 30 min ina 37° C. incubator. Gels were incubated at 37° C. for 24 hours with thefollowing enzymes:

-   -   2 mg (250 U) collagenase    -   8 mg (250 U) α-amylase #1

Rheological testing was conducted on an Anton Paar MCR 302 rheometerusing a 25 mm parallel plate (for solid gels) and 25 mm cone and plate(for completely liquefied gels, i.e., collagen gel treated withcollagenase). All frequency sweeps were conducted at 37° C. and 1%strain (linear viscoelastic region as determined by a strain amplitudesweep). The data are shown in FIG. 1, for the storage and loss modulivs. frequency, and in FIG. 2, for the complex moduli for untreatedcollagen gel, for amylase treated collagen gel and collagenase treatedcollagen gel.

In FIG. 1, storage (G′) and loss (G″) moduli versus frequency arepresented for untreated collagen gel controls, collagen gels treatedwith 250 Units collagenase, and collagen gels treated with 250 Unitsα-amylase #1. The storage modulus represents the solid-like nature andthe loss modulus represents the liquid-like nature of the viscoelasticcollagen gel. There is no significant difference between storage andloss moduli between the untreated collagen gel and the gel treated withα-amylase #1, demonstrating no collagen (protein) gel digestion byα-amylase. The protease collagenase completely liquefied the collagengel, which is demonstrated by the significantly lower storage and lossmoduli.

In FIG. 2, complex modulus (G*) is plotted versus frequency foruntreated collagen gel controls, collagen gels treated with 250 Unitscollagenase, and collagen gels treated with 250 Units α-amylase #1.Complex modulus accounts for the storage modulus (solid-like behavior)and loss modulus (liquid-like behavior), which correlates with the gel'sstiffness. α-Amylase #1 does not digest the collagen gels compared tothe untreated collagen gel control (no statistically significantdifference in complex moduli).

These Figures demonstrate that collagenase, a protease, completelyliquefied the collagen gels within 24 hours, whereas α-amylase #1, aprotein enzyme noted for cleavage of α-linked polysaccharides, such asthose in starch and glycogen, did not digest the collagen gel comparedto the untreated collagen gel control (no statistically significantdifference in storage moduli, loss moduli, or complex moduli). Fromthese Figures, moduli are observed to increase with frequency due to theviscoelastic nature of the polymer (collagen) tested. At highfrequencies, the collagen polymer chains do not have time to relax,resulting in an observed stiffer viscoelastic behavior. The data forcollagen gel treated with collagenase appears noisy due to theliquid-like nature of the resulting digestion of the collagen gel.Collagenase completely degraded and liquefied the collagen gel, and theresulting enzymatically degraded solution had to be evaluated using coneand plate geometry on the rheometer. While the gel was completedliquefied, the resulting solution was still viscoelastic due to thenature of the collagen and peptides remaining in the solution.

The rheology data supports the debridement activity on devitalizedtissue by α-amylase as not being dependent on contamination by aprotease.

Ex Vivo Method for Evaluating Digestion Efficacy of Various Enzymes inSolution

In U.S. Pat. No. 8,119,124, an in vivo (ex vivo) burn wound model wasreported using the skin of young swine because of its similarity to thatof humans. In this invention, frozen pig skin was obtained from CulebraMeat Market, San Antonio, Tex. For each experiment, the pig skin wasboiled in water for 1 min, and then cut into small square pieces. Theboiled pig skin was weighed immediately prior to the application of adebridement formulation. The boiled pig skin was incubated with 1 gramof each debridement formulation for 16 h at 34° C., and wiped gentlywith a piece of paper towel to remove any digested tissue. The remainingtissue was then weighed. As a control, pig skin was treated withoutactive enzyme by the same procedure. The percent digestion of pig skinwas calculated using formula (1):

$\begin{matrix}{{\% \mspace{14mu} {digested}\mspace{14mu} {pig}\mspace{14mu} {skin}} = {\frac{W_{2}\mspace{11mu} {enz}}{W_{1}\mspace{11mu} {enz}*\frac{W_{2}{veh}}{W_{1}\; {veh}}}*100\%}} & (1)\end{matrix}$

whereW₁ enz: weight of pig skin prior to digestion in a formulation thatcontains one or more active enzymes.W₁ veh: weight of pig skin prior to digestion in the same formulationexcept without any active enzymes.W₂ enz: weight of pig skin after digestion in a formulation thatcontains one or more active enzymes.W₂veh: weight of pig skin after digestion in the same formulation exceptwithout any active enzymes.

Ex Vivo Method for Evaluating Digestion Efficacy of α-Amylase Powder

Frozen pig skin was obtained from Culebra Meat Market, San Antonio, Tex.The pig skin was boiled in water for 1 minute, and then cut into 2 cmsquare pieces. The boiled pig skin was weighed immediately prior to theapplication of the amylase powder. α-Amylase #1 or α-amylase #6 wasadded on top of the pig skin. Cotton gauze wetted with saline was placedaround the pig skin, and wrapped by a blue surgical pad secured withElastikon bandage. The wrapped pig skin was then incubated at 37° C. for24 hours. After incubation, the pig skin was removed and wiped gentlywith a paper towel to remove any digested tissue. The remaining tissuewas then weighed. As a control, pig skin was treated without activeenzyme by the same procedure (Table 12). The percent digestion of pigskin was calculated using formula (1) above.

Ex Vivo Pig Skin Digestion Results for α-Amylase #1-7, β-Amylase, andγ-Amylase #1,2, in Solution

In U.S. Pat. No. 8,119,124, an in vivo (ex vivo) burn wound model wasreported using the skin of young swine because of its similarity to thatof humans. In our investigation, several samples of frozen pig skin wereobtained from Culebra Meat Market, San Antonio, Tex., and used as adebridement analog. For each digestion experiment, the pig skin wasboiled in water for 1 minute, and then cut into small square pieces. Theboiled pig skin was weighed immediately prior to the application of adebridement formulation. It was then incubated with 1 gram of eachdebridement formulation for 16 hours at 34° C. in water at pH 7, andwiped gently with a piece of paper towel to remove any digested tissue.The remaining tissue was then weighed. As a control, boiled pig skin wastreated without active enzyme by the same procedure. The percentdigestion of pig skin was calculated using the following formulation:

${\% \mspace{14mu} {digested}\mspace{14mu} {pig}\mspace{14mu} {skin}} = {\frac{W_{2}\; {enz}}{W_{1}\; {enz}*\frac{W_{2}{veh}}{W_{1}\; {veh}}}*100\%}$

whereW₁ enz: weight of pig skin prior to digestion in a formulation thatcontains one or more active enzymes.W₁ veh: weight of pig skin prior to digestion in the same formulationexcept without any active enzymes.W₂ enz: weight of pig skin after digestion in a formulation thatcontains one or more active enzymes.W₂veh: weight of pig skin after digestion in the same formulation exceptwithout any active enzymes.

Digestion Results, Pig Skin

Amylase from different sources, or at different activity levelsexpressed in Units (U), showed different levels of ex vivo debridementactivity (Table 1). α-Amylase from porcine pancreas from both SigmaAldrich (α-amylase #1) and Lee BioSolutions (α-amylase #2, α-amylase #3)showed high debridement efficacy, where 1 gram of formulation contains250 U of enzyme. Surprisingly, the α-amylase (Lee BioSolutions, 0.2%protease) that contained a higher amount of protease impurity (α-amylase#2) had a slightly lower debridement efficacy than the α-amylase thatcontained a lower amount of protease impurity (Lee BioSolutions, 0.05%protease) (α-amylase #3), further supporting the conclusion that amylasedebridement activity is not a result of protease contamination.α-Amylase from human saliva (α-amylase #4) was ineffective in digestingpig skin at the concentration studied. The activity of α-amylase derivedfrom bacteria is highly variable depending on the bacterial strain andthe vendor. The α-amylase from Bacillus licheniformis (α-amylase #5)showed very weak debridement efficacy, whereas the α-amylase fromBacillus subtilis spp. showed high debridement efficacy (α-amylase #6,α-amylase #7), particularly at an activity of 250,000 Units of enzyme in1 gram of formulation. β-Amylase showed some activity in digesting pigskin based upon its ability to act upon the same substrates as α-amylasebut with a different catalytic mechanism, whereas γ-amylase #1 and #2were ineffective in this debridement model.

TABLE 1 Ex vivo debridement efficacy of amylases from various sources onboiled pig skin in water formulation formulation formulation ratio (wt%) (Units) % digested H₂O/α-amylase #1 99.2/0.8 1 g/250 U 96.30%H₂O/α-amylase #2 99.891/0.109 1 g/250 U 96.60% H₂O/α-amylase #399.88/0.12 1 g/250 U 97.90% H₂O/α-amylase #4 99.8/0.2 1 g/250 U  4.30%H₂O/α-amylase #5 99.95 g/0.05  1 g/250 U    0% H₂O/α-amylase #5 99.9/0.11 g/500 U 13.50% H₂O/α-amylase #6 99.9966/0.0033 1 g/250 U 17.50%H₂O/α-amylase #6 99.966/0.034 1 g/2,500 U 70.80% H₂O/α-amylase #696.6/3.4 1 g/250,000 U  100% H₂O/α-amylase #7 99.9803/0.0197 1 g/250 U15.00% H₂O/α-amylase #7 99.803/0.197 1 g/2,500 U 77.60% H₂O/α-amylase #7 80.3/19.7 1 g/250,000 U  100% H₂O/β-amylase 99.4/0.6 1 g/250 U 18.60%H₂O/β-amylase 94/6 1 g/2500 U 38.90% H₂O/γ-amylase #1 99.8/0.2 1 g/250 U   0% H₂O/γ-amylase #1 99/1 1 g/1,250 U    0% H₂O/γ-amylase #1 98/2 1g/2,500 U    0% H₂O/γ-amylase #2 99.4/0.6 1 g/250 U    0%

In Table 2 are listed the effects of various additives and excipients onthe debridement efficacy of α-amylase #1 with boiled pig skin. Includedis a DPBS buffered solution, a formulation in mineral oil, a formulationof a hydrophilic, a viscosity increasing polymer, HPMC, in DPBS, and aformulation with a viscosity increasing cationic polymer, Polymer JR(cationic hydroxyethylcellulose). The results are compared to α-amylase#1 in water at pH 7. Each formulation contained the same amount ofα-amylase #1, namely 250 Units. It is seen that the formulationconsistency affected the amylase debridement efficacy. The addition ofDPBS buffer appeared to enhance the debridement efficacy compared toα-amylase in water, whereas mineral oil greatly decreased thedebridement efficacy, presumably because of the lack of water forenzymatic solubility and activity; the added water-soluble polymers ofneutral cellulose-based HPMC in DPBS and cationic cellulose-basedPolymer JR in water somewhat decreased the debridement efficacy,possibly because of a lower content of water in their formulationscompared to α-amylase #1 by itself in water.

TABLE 2 Ex vivo debridement efficacy of α-amylase #1 in variousformulations on boiled pig skin formulation formulation formulationratio (wt %) (Units) % digested H₂O/α-amylase #1 99.2/0.8 1 g/250 U96.30% DPBS/α-amylase #1 99.2/0.8 1 g/250 U  100% Mineral oil/α-99.2/0.8 1 g/250 U 62.90% amylase #1 HPMC/DPBS/α- 5/94.2 g/0.8 0.05g/0.95 80.10% amylase #1 g/250 U Polymer JR-30M/H₂O/α- 5/94.2 g/0.8 0.05g/0.95 84.30% amylase #1 g/250 U

In Table 3 are described combinations of α-amylase and I-amylase usingbacterial α-amylase from Bacillus subtilis (α-amylase #6) in conjunctionwith plant derived I-amylase. In this model, the greater amount ofI-amylase appeared to hinder the overall digestion of the boiled pigskin.

TABLE 3 Ex vivo debridement efficacy of boiled pig skin by α-amylase #6with β-amylase in water formulation formulation formulation ratio (wt %)(Units) % digested H₂O/α-amylase #6 99.66/0.34 1 g/250 U 79.20%H₂O/α-amylase 96.6/0.34/3.16 1 g/250 U/131 U 53.70% #6/β-amylaseH₂O/α-amylase 99.32/0.34/0.34 1 g/250 U/14 U 72.30% #6/β-amylase

In Table 4, the addition of another non-proteolytic hydrolytic enzyme,lipase, to α-amylase #3 on the digestion of boiled pig skin is given,using amylase contents of 100 wt %, 90 wt %, and 80 wt % to lipase.Lipase is noted for its ability to catalyze the hydrolysis of esterbonds in lipids (triglycerides, fats, and oils). There are multiplesources of lipase as it is produced by the pancreas, liver, intestine,tongue, stomach, and many other cells, as well as the seeds of plants.The addition of lipase to α-amylase could be a major adjunct indebridement wherein fatty tissue is involved. In the pig model studied,the % boiled pig skin digested was relatively constant for the threesamples studied, using a total enzyme concentration of 0.2 wt %, eventhough the amount of amylase #3 decreased with increasing lipasecontent, indicating that both non-proteolytic enzymes contributed to thedigestion of boiled pig skin.

TABLE 4 Ex vivo debridement efficacy of boiled pig skin by α-amylase #3with lipase #2 in water formulation formulation formulation ratio (wt %)(Units) % digested H₂O/α-amylase #3 99.8/0.2 1 g/420 U 83.90%H₂O/α-amylase 99.8/0.18/0.02 1 g/378 84.10% #3/lipase #2 U/72 UH₂O/α-amylase 99.8/0.16/0.04 1 g/336 81.20% #3/lipase #2 U/144 U

In Table 5 are given the results of pig skin digested by α-amylase #1 inwater with the keratolytic agent urea, with chlorophyllin for reducinglocal inflammation, promoting healing, and controlling odor, and with acombination of urea and chlorophyllin. All solutions with chlorophyllinwere green colored. It is seen that a different source of pig skin,compared to that of Table 1, done under similar conditions, had areduced digestion level. The addition of urea appeared to increase thedigestion efficacy by α-amylase, while the addition of twoconcentrations of chlorophyllin appeared to decrease the amylaseefficacy. A combination of the three ingredients gave a digestionefficacy greater than chlorophyllin and α-amylase, and less than that ofurea and amylase, or α-amylase by itself.

TABLE 5 Ex vivo debridement efficacy of boiled pig skin by α- amylase #1with urea and chlorophyllin in water formulation formulation formulationratio (wt %) (Units) % digested H₂O/α-amylase #1 99.2/0.8 1 g/250 U78.60% H₂O/urea/α-amylase #1 89.2/10/0.8 1 g/250 U 84.90%H₂O/chlorophyllin/α- 98.7/0.5/0.8 1 g/250 U 54.80% amylase #1H₂O/chlorophyllin/α- 98.2/1/0.8 1 g/250 U 59.30% amylase #1H₂O/urea/chlorophyllin/α- 88.7/10/0.5/0.8 1 g/250 U 67.20% amylase #1

Necrotic tissue is susceptible to bacterial infection, which furtherimpedes wound healing, and may induce sepsis in severe cases. Because ofthe possibly of infection in necrotic wounds and damaged and irritatedtissue, addition of a biological agent that hinders or eradicatesmicroorganisms is desired. In this regard, we investigated twoantimicrobial biguanides used in wound care, poly(hexamethylenebiguanide hydrochloride) (PHMB) and chlorhexidine digluconate (CHG).Table 6 is a comparison of three formulations of aqueous solutions ofPHMB with α-amylase #6 and CHG with α-amylase #6, all at the same wt %of α-amylase #6. For the PHMB-based solutions, these activities wereanalogous or slightly greater than that of α-amylase by itself, althoughthe higher PHMB level of 0.15 wt % (1500 ppm) may have slightlydecreased the amylase activity. The use of CHG, however, appeared todecrease markedly the amylase digestion ability.

TABLE 6 Ex vivo debridement of pig skin by α-amylase #6 with PHMB andCHG in water formulation formulation formulation ratio (wt %) (Units) %digested H₂O/α-amylase #6 99.66/0.34 1 g/25,000 U 83.90%H₂O/PHMB/α-amylase #6 99.51/0.15/0.34 1 g/25,000 U 81.30%H₂O/PHMB/α-amylase #6 99.56/0.1/0.34 1 g/25,000 U 84.90%H₂O/PHMB/α-amylase #6 99.61/0.05/0.34 1 g/25,000 U 86.80%H₂O/CHG/α-amylase #6 97.66/2/0.34 1 g/25,000 U 60.50%

Ex Vivo Digestion Results for α-Amylase #1 and α-Amylase #6, Rat Skin

Freshly excised rat skin tissue was obtained from two adult rats 9 and11 months of age from the Laboratory Animal Resources Center at theUniversity of Texas at San Antonio, San Antonio, Tex., under an IACUCapproved protocol. A large piece of skin was retrieved from the back ofeach male Sprague-Dawley rat. Half of the skin was cut and boiled inwater for 60 seconds. Both the boiled and unboiled halves of the skinwere cut into smaller pieces and trimmed so that each small pieceweighed from 0.23 g to 0.25 g. One small piece of skin was soaked in 1gram of each debridement formulation and incubated at 34° C. to debridefor 24 hours. After the 24-hour incubation, the skin was removed fromthe debridement formulation. The debrided skin (soft and mushy tissue)was gently wiped with paper towels, and the remaining non-debrided skinwas weighed. The percent of debrided skin was then calculated based onthe weight before and after the debridement procedure.

Tables 7 and 8 present the debridement results of freshly excised ratskin from that which was debrided under unboiled and boiled conditions,respectively, using the amylolytic enzymes α-amylase #1 and α-amylase #6as well as the protease enzymes of bromelain and collagenase, all inwater at pH 7. For the unboiled rat skin (Table 7), it is seen that thetwo protease enzymes were more effective than either amylase, withcollagenase being the most effective in digestion of rat skin at 79%.However, for boiled rat skin (Table 8), which is more analogous todevitalized, necrotic tissue, the amylolytic enzymes were comparable tothe proteolytic enzymes in their ability to digest rat skin.

TABLE 7 Ex vivo debridement of unboiled rat skin by α-amylase,bromelain, and collagenase in water formulation formulation formulationratio (wt %) (Units) % digested H₂O/α-amylase #1 99.2/0.8 1 g/250 U31.80% H₂O/α-amylase #6 99.66/0.34 1 g/25,000 U 29.80% H₂O/bromelain 90/10 1 g/250 U 51.90% H₂O/collagenase 99.8/0.2 1 g/250 U   79%

TABLE 8 Ex vivo debridement of boiled rat skin by α-amylase, bromelain,and collagenase in water formulation formulation formulation ratio (wt%) (Units) % digested H₂O/α-amylase #1 99.2/0.8 1 g/250 U 86.80%H₂O/α-amylase #6 99.66/0.34 1 g/25,000 U 90.40% H₂O/bromelain  90/10 1g/250 U 92.90% H₂O/collagenase 99.8/0.2 1 g/250 U   86%

If necrotic tissue is infected with a microbial biofilm, potentiallyfurther covering the necrotic tissue with additional polysaccharides (acomponent of an extracellular polymeric substance, sometimes referred toas slime) emanating from the biofilm microorganisms, which may bepotentially bacterial in origin, removal or reduction of the biofilm mayexpedite the removal of necrotic or devitalized tissue. Tables 9 and 10give the results of using an aqueous antimicrobial composition (AC) atpH 5.5 that has demonstrated efficacy in biofilm reduction (U.S. Pat.No. 8,829,053). The antimicrobial composition contains a polymericbiguanide antimicrobial agent of PHMB, EDTA as a chelating and pHstabilizing agent, HPMC as a viscosity increasing polymer, a neutralsurfactant of P407 for cleansing, and a combination of 2-ethylhexylglycerin and 1,2-octanediol for emollient and antimicrobial properties.With the rat skin in an unboiled condition in the antimicrobialcomposition (Table 9), neither α-amylase #1 nor α-amylase #6 was aseffective in digestion relative to that displayed by the same enzymesfor unboiled rat skin in water (Table 7). Although bromelain in theantimicrobial composition was more effective than either amylase withthe unboiled rat skin (Table 9), its digestion effect is less than thatdemonstrated for the digestion of the rat skin in water (Table 7). Whilecollagenase was very effective for both unboiled and boiled rat skindigestion in water (Tables 7 and 8), for the unboiled rat skin in thepresence of the aqueous antimicrobial composition, its effect indigestion was negligible (Table 9). This effect also occurred forcollagenase with the boiled rat skin in the antimicrobial composition(Table 10), while α-amylase #1, α-amylase #6, and bromelain were highlyeffective (Table 10). Since the antimicrobial composition contains themetal ion chelating agent EDTA, this may have inactivated collagenase,which is a metalloproteinase (zinc endopeptidase). The calcium complexof amylase appears not to be affected by EDTA chelation, and theantimicrobial compositions of α-amylase would be an effectivenon-proteolytic treatment of necrotic tissue infected with microbialbiofilm. The low debridement efficacy of α-amylase #1 and α-amylase #6on unboiled, freshly excised rat skin in the antimicrobial composition(Table 9) also supports the amylolytic debridement composition beinghighly specific to devitalized tissue and not toward the surroundingliving tissue.

TABLE 9 Ex vivo debridement of unboiled rat skin by α-amylase #1,α-amylase #6, bromelain, and collagenase in an antimicrobial compositionformulation formulation formulation ratio (wt %) (Units) % digestedAC/α-amylase #1 99.2/0.8 1 g/250 U 6.00% AC/α-amylase #6 99.66/0.34 1g/250 U   0% AC/Bromelain  90/10 1 g/250 U 18.20%  AC/collagenase99.8/0.2 1 g/250 U 4.70%

TABLE 10 Ex vivo debridement of boiled rat skin by α-amylase #1,α-amylase #6, bromelain, and collagenase in an antimicrobial compositionformulation formulation formulation ratio (wt %) (Units) % digestedAC/α-amylase #1 99.2/0.8 1 g/250 U 96.50%  AC/α-amylase #6 99.66/0.34 1g/250 U 100% AC/bromelain  90/10 1 g/250 U 100% AC/collagenase 99.8/0.21 g/250 U  0%

Excipients

In addition to water and buffered solutions (Table 2), as well as beingutilized in powder form (Table 12), the amylases can be mixed withseveral excipients, including hydrophobic hydrocarbons of petrolatum andmineral oil, hydrophilic —OH containing alcohols of glycerin and PEG400, and the amphiphilic liquid PEG-12 Dimethicone, a siliconepolyether. In each case, α-amylase #1 could be dispersed in theexcipient (Table 11). Other excipients could include various water-basedbuffers ranging in pH from 5.0-7.5, surfactants, silicones, polyethercopolymers, polyoxyethylene ethers, vegetable and plant fats and oils,essential oils, hydrophilic and hydrophobic alcohols, vitamins,monoglycerides, laurate esters, myristate esters, palmitate esters, andstearate esters, preferably in liquid, gel, or paste form, combinationsthereof, and the like. In some embodiments, excipients can be present inan amount ranging from 0% to 99.9 wt % based on the weight of thedebridement composition.

TABLE 11 Excipients for α-amylase #1 formulation formulation formulationratio (wt %) (Units) petrolatum/α-amylase #1 99.2/0.8 1 g/250 U mineraloil/α-amylase #1 99.2/0.8 1 g/250 U glycerin/α-amylase #1 99.2/0.8 1g/250 U PEG 400/α-amylase #1 99.2/0.8 1 g/250 U DC 193/α-amylase #199.2/0.8 1 g/250 U

Effective α-Amylase Concentration

To determine the amount of α-amylase needed for debridement of necrotictissue, a study was conducted to measure debridement efficacy (e.g., wt% digested) verses α-amylase concentration. The results in FIG. 3 showsthe concentration of α-amylase #6 versus the percent by weight of boiledpig skin digested in one hour at room temperature. The bell-shaped curveshows that debridement activity occurs at a maximum amylaseconcentration in the range of between 13.6 to 27.2 wt %, though theefficacy is 10 wt % or greater over the entire range from 3.4% to 54.5%α-amylase.

Ex vivo Digestion Results, α-Amylase Powder

α-Amylase #1 and #6 powders demonstrated high levels of ex vivodebridement activity. In 24 hours, wherein 71.94% of pig skin wasdigested by α-amylase #1 powder and 99.14% of pig skin was digested byα-amylase #6 powder (Table 12).

TABLE 12 Ex vivo debridement efficacy of boiled pig skin by α-amylasepowder weight of amylase Amylase powder powder (grams) % digestedα-amylase #1 0.01 71.94% α-amylase #6 0.1 99.14%

The amylase powder can be admixed with other powders that function asexcipients including powdered cellulose, microcrystalline cellulose,magnesium stearate, polyvinylpyrrolidone and copolymers thereof,crosslinked polyvinylpyrrolidone, polysaccharides, poly(vinyl alcohol),poly(vinyl alcohol-co-vinyl acetate), polyglycerol, stearic acid,stearyl alcohol, cetyl alcohol, gelatin, maltodextrin, calcium alginate,glycerol monostearate, glyceryl distearate, glyceryl tristearate,glyceryl palmitostearate, poloxomers, biodegradable polymers, and thelike.

Ex Vivo Pig Skin Digestion Results, Fungal Amylases, in Water and Cream

Utilizing procedures similar to those above, α-amylase #8a and α-amylase#8b from the fungus Aspergillus oryzae showed high levels of ex vivodebridement activity, in either water solution or a cream basedformulation (Table 13).

TABLE 13 Ex vivo debridement efficacy of boiled pig skin by α-amylase#8a, #8B formulation formulation ratio (wt %) % digested H₂O/α-amylase#8a 96.6/3.4 72.66% H₂O/α-amylase #8b  90/10 75.92%H₂O/petrolatum/SA/PG/α- 87.96/10/10/5/5/ 84.10% amylase #8b/P40S/SM/PHMB0.5/0.5/0.02

In Vivo Porcine Debridement Efficacy by Fungal α-Amylase #8a

Debridement efficacy of α-amylase #8a was tested in vivo on porcine burnwounds. The porcine burn wound model was chosen for the in vivo studybecause this model is well established for testing the debridementefficacy of commercial products, and the wound size and depth can beeasily controlled (Shi, L. et al., “Wound Repair Regen., 2009,17(6):853-62). α-Amylase #8a was tested in vivo at a dosage of 10 wt %in a cream formulation.

The debridement cream was made as follows:

Cream Base

60 grams of Part A were mixed with all ingredients in Part B and thenheated to 65° C. in a water bath to melt all ingredients. The mixturewas then blended by an overhead stirrer at 600 rpm for 10 minutes. Whilestirring, the heat source was removed and the water bath was let coolslowly to room temperature.

Part A

1.52 grams of PHMB (Cosmocil CQ) was dissolved in 158.48 grams of water

Part B

White Petrolatum: 60 gramsStearyl Alcohol: 60 gramsSorbitan monostearate: 3 gramsPolyoxyl 40 Stearate: 3 gramsPropylene Glycol: 30 grams

Fungal Amylase Solution

An aqueous fungal amylase solution was prepared from 60 grams ofα-amylase #8b dissolved in 108 grams of water, and filtered with avacuum filter (pore size 0.22 μm).

Fungal Amylase Cream

The amylase cream was prepared from 84 grams of amylase solution thatwas slowly added to 216 gram of cream base while stirring at roomtemperature until the mixture became a homogeneous brown cream with novisible particles (Table 14).

TABLE 14 Formulation for In vivo debridement with α-amylase #8bformulation formulation ratio (wt %) H₂O/petrolatum/SA/PG/α-37.96/20/20/10/10/1/1/0.04 amylase #8b/P40S/SM/PHMB

An in vivo pig study was conducted at Surpass Inc. (Mountain View,Calif.). One female Yorkshire pig weighing 40-60 kg was used in thestudy. At day 0, ten (10) deep partial thickness burn wounds (20 mmdiameter) were created on one side of the pig using a temperaturecontrolled brass rod with a diameter of 2 cm. The brass rod was heatedto 200° C. and placed on the skin for 80 seconds. The temperature wascontrolled and remained at 200° C. throughout the burn. No surgicaldebridement occurred after burn.

Fungal α-amylase cream from Aspergillus oryzae (#8b) was applied to theburn eschar on day 0, day 1, day 2, and day 3, and the animal waseuthanized on day 4.

In applying the debridement treatment, 0.5 ml of each test material wasplaced on the wound site using a needleless syringe. The wounds werethen covered with Telfa™ dressing moistened with sterile saline andsecured with Transpore® tape. The pig was then wrapped with a bluesurgical pad, and an Elastikon bandage was wrapped around the pig tohold the surgical pad in place.

Visual clinical assessments of the wound areas were performed andsemi-quantitative scores were given for eschar (1=no eschar; 2=escharcovering ≦25%; 3=eschar covering 26-50%; 4=eschar covering 51-75%;5=eschar covering 76-100%).

By day 2, areas of wounds that were covered by the debrider were fullydebrided, demonstrating the debridement efficacy of fungal Aspergillusoryzae α-amylase. The eschar scores for Fungal α-amylase cream fromAspergillus oryzae (#8b) were as follows:

5.0±0.0 at day 0

5.0±0.0 at day 1

1.0±0.0 at day 2

1.0±0.0 at day 3

1.0±0.0 at day 4.

While the above specification contains many specifics, these should notbe construed as limitations on the scope of the invention, but rather asexamples of preferred embodiments thereof. Many other variations arepossible. Accordingly, the scope of the invention should be determinednot by the embodiments illustrated, but by the appended claims and theirlegal equivalents.

1. A tissue debridement composition comprising: 0.1 to 99.5 wt % of anamylase component and optionally a proteolytic enzyme component, whereina ratio of the amylase component to the proteolytic enzyme component isat least 10:1 by weight; and at least one pharmaceutically orcosmetically acceptable carrier or excipient in an amount of up to 99.9wt %, wherein the percentages are based on a total weight of thedebridement composition, wherein said amylase component comprises atleast 80% by weight of α-amylase from Aspergillus oryzae, and whereinthe debridement composition is able to debride devitalized or necrotictissue.
 2. The composition according to claim 1, wherein said amylasecomponent comprises up to 20% by weight of an amylase selected from agroup consisting of β-amylase, γ-amylase, and combinations thereof. 3.The composition according to claim 1, further comprising up to 20% byweight of hydrolytic enzymes selected from a group consisting ofproteases, chondroitinases, hyaluronidases, lipases, glycosidases,heparanases, dermatanases, pullulanases, N-acetylglucosaminidase,lactases, phospholipases, transglycosylases, esterases, thioesterhydrolyases, sulfatases, escharases, nucleases, phosphatases,phosphodiesterases, mannanases, mannosidases, isoamylases, lyases,inulinases, keratinases, tannases, pentosanases, glucanases,arabinosidases, pectinases, cellulases, chitinases, xylanases,cutinases, pectate lyases, hemicellulases, and combinations thereof,wherein the percentage is based on the total weight of the amylasecomponent and the hydrolytic enzymes.
 4. The composition according toclaim 1, further comprising up to 20% by weight of other enzymesselected from a group consisting of oxidases, peroxidases, glucoseoxidases, catalases, oxidoreductases, phenoloxidases, laccases,lipoxygenases, isomerases, and ligninases, wherein the percentage isbased on the total weight of the amylase component and the otherenzymes.
 5. The composition according to claim 1, further comprising atleast one polymeric biguanide in an amount ranging from 0.01 weightpercent to 1.0 weight percent based on the weight of the debridementcomposition.
 6. The composition according to claim 5, wherein saidpolymeric biguanide comprises poly(hexamethylene biguanide) and itssalts.
 7. The composition according to claim 1, further comprising awater-soluble polymer at a concentration from 0.01 weight % to 50 weight% based on the weight of the debridement composition.
 8. The compositionaccording to claim 1, further comprising a chelating agent at aconcentration from 0.01 weight % to 1 weight % based on a weight of thedebridement composition.
 9. The composition according to claim 1,further comprising a monoalkyl glycol selected from a group consistingof 1,2-propanediol (propylene glycol), 1,2-butanediol, 1,2-pentanediol,1,2-hexanediol, 1,2-heptanediol, 1,2-octanediol (caprylyl glycol),1,2-nonanediol, 1,2-decanediol, 1,2-undecanediol, 1,2-dodecanediol,1,2-tridecanediol, 1,2-tetradecanediol, 1,2-pentadecanediol,1,2-hexadecanediol, 1,2-heptadecanediol, 1,2-octadecanediol,2-methyl-2,4-pentanediol, 1,3-butanediol, diethylene glycol, triethyleneglycol, glycol bis(hydroxyethyl) ether, and combinations thereof. 10.The composition according to claim 1, further comprising a glycerolalkyl ether selected from a group consisting of 1-O-heptylglycerol,1-O-octylglycerol, 1-O-nonylglycerol, 1-O-decylglycerol,1-O-undecylglycerol, 1-O-dodecylglycerol, 1-O-tridecylglycerol,1-O-tetradecylglycerol, 1-O-pentadecylglycerol, 1-O-hexadecylglycerol(chimyl alcohol), 1-O-heptadecylglycerol, 1-O-octadecylglycerol (batylalcohol), 1-O-octadec-9-enyl glycerol (selachyl alcohol), glycerol1-(2-ethylhexyl) ether, 2-ethylhexyl glycerin, glycerol 1-heptyl ether,glycerol 1-octyl ether, glycerol 1-decyl ether, and glycerol 1-dodecylether, glycerol 1-tridecyl ether, glycerol 1-tetradecyl ether, glycerol1-pentadecyl ether, glycerol 1-hexadecyl ether, glycerol 1-octadecylether, and combinations thereof.
 11. The composition according to claim1, further comprising at least one polymeric biguanide in an amountranging from 0.01 weight % to 1.0 weight % based on the weight of thedibridement composition, and a vicinal diol component selected from agroup consisting of a monoalkyl glycol and a monoalkyl glycerol at aconcentration of 0.05 weight % to 4 weight %, based on a weight of thedebridement composition.
 12. The composition according to claim 1,further comprising at least one analgesic agent, anesthetic agent,neuropathic pain agent, or a combination thereof.
 13. The compositionaccording to claim 12, wherein the at least one analgesic agent,anesthetic agent, or neuropathic pain agent is selected from a groupconsisting of lidocaine, capsaicin, benzocaine, tetracaine, prilocaine,bupivacaine, levobupivacaine, procaine, carbocaine, etidocaine,mepivacaine, nortripylene, amitriptyline, pregabalin, diclofenac,fentanyl, gabapentin, non-steroidal anti-inflammatory agents,salicylates, and combinations thereof.
 14. The composition according toclaim 1, wherein the debridement composition has a form selected from agroup consisting of a powder, aqueous solution, organic liquid solution,silicone, gel, cream, film, latex, aerosol, slurry, paste, ointment andfoam.
 15. The composition according to claim 1, wherein said debridementcomposition is adsorbed on or in a dressing or a natural or syntheticfiber, mesh, hydrocolloid, alginate, hydrogel, semipermeable film,permeable film, or a natural or synthetic polymer.
 16. The compositionaccording to claim 1, further comprising a buffering agent.
 17. Thecomposition according to claim 1, wherein the amylase component ispresent in an amount ranging from 2 to 99.5 wt-% based on the totalweight of the debridement composition.
 18. A kit comprising: a tissuedebridement composition according to claim 1; and instructions fordebridement of devitalized tissue.
 19. The kit according to claim 18,wherein said instructions comprise contacting the tissue debridementcomposition with an area of skin in need of debridement.
 20. A method ofdebridement of devitalized tissue comprising contacting a tissuedebridement composition according to claim 1 with an area of skin inneed of debridement.
 21. The method of claim 20, wherein said contactingstep is repeated at least once a day.
 22. The method of claim 20,further comprising removing debrided tissue from said area of skin. 23.A composition for removal of devitalized or necrotic tissue comprising:0.01 to 100 wt % of solid amylase and, optionally, a dry proteolyticenzyme component, wherein a ratio of the amylase component to theproteolytic enzyme component is at least 10:1 by weight; and optionallya pharmaceutically or cosmetically acceptable carrier or excipient in anamount of up to 99.99 wt %, wherein the percentages are based on a totalweight of the solid composition, wherein said amylase componentcomprises at least 80% by weight of α-amylase from a source selectedfrom a group consisting of porcine pancreas, Bacillus subtilis, andAspergillus oryzae.